Use of interleukin-4 antagonists and compositions thereof

ABSTRACT

Methods for treating medical conditions induced by interleukin-4 involve administering an IL-4 antagonist to a patient afflicted with such a condition. Suitable IL-4 antagonists include, but are not limited to, IL-4 receptors (such as a soluble human IL-4 receptor), antibodies that bind IL-4, antibodies that bind IL-4R, IL-4 muteins that bind to IL-4R but do not induce a biological response, molecules that inhibit IL-4-induced signal transduction, and other compounds that inhibit a biological effect that results from the binding of IL-4 to a cell surface IL-4R. Particular antibodies provided herein include human monoclonal antibodies generated by procedures involving immunization of transgenic mice. Such human antibodies may be raised against human IL-4 receptor. Certain of the antibodies inhibit both IL-4-induced and IL-13-induced biological activities.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/893,101, filed Feb. 9, 2018, which is a continuation of U.S.application Ser. No. 15/414,447, filed Jan. 24, 2017, abandoned, whichis a divisional of U.S. application Ser. No. 14/175,943, filed Feb. 7,2014, now U.S. Pat. No. 9,587,026, which is a divisional of U.S.application Ser. No. 12/829,231, filed Jul. 1, 2010, now U.S. Pat. No.8,679,487, which is a continuation of U.S. application Ser. No.12/291,702, filed Nov. 13, 2008, abandoned, which is a continuation ofU.S. application Ser. No. 11/588,696, filed Oct. 27, 2006, now U.S. Pat.No. 7,465,450, which is a divisional of U.S. application Ser. No.10/324,493, filed Dec. 19, 2002, now U.S. Pat. No. 7,186,809, which is acontinuation of U.S. application Ser. No. 09/847,816, filed May 1, 2001,abandoned. The above-identified applications are incorporated herein byreference.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format via EFS-Web. The Sequence Listing is provided as atext file entitled 3005-US-CNT5_SequenceListing.txt, created Oct. 3,2018, which is 55,606 bytes in size. The information in the electronicformat of the Sequence Listing is incorporated herein by reference inits entirety.

BACKGROUND OF THE INVENTION

Interleukin-4 (IL-4), previously known as B cell stimulating factor, orBSF-1, was originally characterized by its ability to stimulate theproliferation of B cells in response to low concentrations of antibodiesdirected to surface immunoglobulin. IL-4 has been shown to possess a farbroader spectrum of biological activities, including growthco-stimulation of T cells, mast cells, granulocytes, megakaryocytes, anderythrocytes. In addition, IL-4 stimulates the proliferation of severalIL-2- and IL-3-dependent cell lines, induces the expression of class IImajor histocompatibility complex molecules on resting B cells, andenhances the secretion of IgE and IgG1 isotypes by stimulated B cells.IL-4 is associated with a TH2-type immune response, being one of thecytokines secreted by TH2 cells.

Murine and human IL-4 have been identified and characterized, includingcloning of IL-4 cDNAs and determination of the nucleotide and encodedamino acid sequences. (See Yokota et al., Proc. Natl. Acad. Sci. USA83:5894, 1986; Noma et al., Nature 319:640, 1986; Grabstein et al., J.Exp. Med. 163:1405, 1986; and U.S. Pat. No. 5,017,691.)

IL-4 binds to particular cell surface receptors, which results intransduction of a biological signal to cells such as various immuneeffector cells. IL-4 receptors are described, and DNA and amino acidsequence information presented, in Mosley et al., Cell 59:335-348, Oct.20, 1989 (murine IL-4R); Idzerda et al., J. Exp. Med. 171:861-873, March1990 (human IL-4R); and U.S. Pat. No. 5,599,905. The IL-4 receptordescribed in these publications is sometimes referred to as IL-4Rα.

Other proteins have been reported to be associated with IL-4Rα on somecell types, and to be components of multi-subunit IL-4 receptorcomplexes. One such subunit is IL-2Rγ, also known as IL-2Rγ_(c). (Seethe discussion of IL-4R complexes in Sato et al., Current Opinion inCell Biology, 6:174-179, 1994.) IL-4Rα has been reported to be acomponent of certain multi-subunit IL-13 receptor complexes (Zurawski etal., J. Biol. Chem. 270 (23), 13869, 1995; de Vries, J. Allergy Clin.Immunol. 102(2):165, August 1998; and Callard et al. Immunology Today,17(3):108, March 1996).

IL-4 has been implicated in a number of disorders, examples of which areallergy and asthma. Studies of biological properties of IL-4 continue,in an effort to identify additional activities associated with thispleiotrophic cytokine, and to elucidate the role IL-4 may play invarious biological processes and diseases.

SUMMARY OF THE INVENTION

The present invention provides methods for treating certain conditionsinduced by IL-4, comprising administering an IL-4 antagonist to apatient afflicted with such a condition. Also provided are compositionsfor use in such methods, comprising an effective amount of an IL-4antagonist and a suitable diluent, excipient, or carrier. EndogenousIL-4 may be contacted with an IL-4 antagonist in alternative methods,such as those involving ex vivo procedures.

Among the conditions to be treated in accordance with the presentinvention are septic arthritis, dermatitis herpetiformis, chronicidiopathic urticaria, ulcerative colitis, scleroderma, hypertrophicscarring, Whipple's Disease, benign prostate hyperplasia, lung disordersin which IL-4 plays a role, conditions in which IL-4-induced epithelialbarrier disruption plays a role, disorders of the digestive system inwhich IL-4 plays a role, allergic reactions to medication, Kawasakidisease, sickle cell crisis, Churg-Strauss syndrome, Grave's disease,pre-eclampsia, Sjogren's syndrome, autoimmune lymphoproliferativesyndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmuneuveitis, tuberculosis, and nephrosis. IL-4 antagonists also find use asadjuvants to allergy immunotherapy and as vaccine adjuvants.

IL-4 antagonists include, but are not limited to, IL-4 receptors(IL-4R), antibodies that bind IL-4, antibodies that bind IL-4R, IL-4muteins that bind to IL-4R but do not induce a biological response,molecules that inhibit IL-4-induced signal transduction, and othercompounds that inhibit a biological effect that results from the bindingof IL-4 to a cell surface IL-4R.

Examples of IL-4 receptors are soluble forms of the human IL-4 receptorof SEQ ID NO:2. Particular antibodies provided herein include humanmonoclonal antibodies generated by procedures involving immunization oftransgenic mice. Such human antibodies may be directed against humanIL-4 receptor, for example.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C present the nucleotide sequence of the coding region of ahuman IL-4 receptor cDNA. The amino acid sequence encoded by the cDNA ispresented as well. The cDNA clone was isolated from a cDNA libraryderived from a human T cell line T22. The encoded protein comprises(from N- to C-terminus) an N-terminal signal peptide, followed by anextracellular domain, a transmembrane region (underlined), and acytoplasmic domain, as discussed further below. The DNA and amino acidsequences of FIGS. 1A to 1C are also presented in SEQ ID NOS:1 and 2,respectively.

FIGS. 2A-2C depict targeted insertion of a neo cassette into the Sma Isite of the μ1 exon. The construct was employed in generating transgenicmice, as described in Example 2. FIG. 2A is a schematic diagram of thegenomic structure of the μ locus. The filled boxes represent the pexons. FIG. 2B is a schematic diagram of the CmD targeting vector. Thedotted lines denote those genomic μ sequences included in the construct.Plasmid sequences are not shown. FIG. 2C is a schematic diagram of thetargeted μ locus in which the neo cassette has been inserted into μ1.

FIGS. 3A and 3B present the nucleotide sequence of a vector designatedpGP1k, as described in Example 3 below. This nucleotide sequence also ispresented in SEQ ID NO:4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for treating certain conditionsinduced by IL-4, and for inhibiting biological activities ofinterleukin-4 (IL-4) in vivo. One method comprises administering an IL-4antagonist to a patient afflicted with such a condition. Compositionsfor use in such methods for treating IL-4-induced conditions also areprovided.

Among the conditions to be treated in accordance with the presentinvention are septic/reactive arthritis, dermatitis herpetiformis,chronic idiopathic urticaria, scleroderma, hypertrophic scarring,Whipple's Disease, benign prostate hyperplasia, lung disorders in whichIL-4 plays a role, conditions in which IL-4-induced epithelial barrierdisruption plays a role, disorders of the digestive system in which IL-4plays a role, including inflammatory bowel disease and otherinflammatory conditions in the gastrointestinal tract, allergicreactions to medication, Kawasaki disease, sickle cell disease(including sickle cell crisis), Churg-Strauss syndrome, Grave's disease,pre-eclampsia, Sjogren's syndrome, autoimmune lymphoproliferativesyndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmuneuveitis, tuberculosis, and nephrosis, as described in more detail below.IL-4 antagonists also find use as adjuvants to allergy immunotherapy andas vaccine adjuvants.

IL-4 antagonists that may be employed include those compounds thatinhibit a biological activity of IL-4. Biological activity(ies) of IL-4that are inhibited by an antagonist in accordance with methods providedherein are activities that play a role in the particular disease to betreated.

Suitable antagonists include, but are not limited to, IL-4 receptors(IL-4R), antibodies that bind IL-4, antibodies that bind IL-4R, IL-4muteins that bind to IL-4R but do not induce biological responses,molecules that inhibit IL-4-induced signal transduction, and othercompounds that inhibit a biological effect that results from the bindingof IL-4 to a cell surface IL-4R. Examples of such IL-4 antagonists aredescribed in more detail below. Particular embodiments of the inventionare directed to novel antibodies, polypeptides, and nucleic acidmolecules, as described below. Antibodies provided herein include, butare not limited to, human monoclonal antibodies that bind to human IL-4receptor, and that function as antagonists of both IL-4 and IL-13.

Indications

The present invention provides methods comprising administering an IL-4antagonist to a patient afflicted with any of a number of conditionsinduced by IL-4. IL-4-induced conditions include conditions caused orexacerbated, directly or indirectly, by IL-4. Other factors or cytokinesalso may play a role in such conditions, but IL-4 induces or mediatesthe condition to some degree, i.e., at least in part.

The biological activities of IL-4 are mediated through binding tospecific cell surface receptors, referred to as interleukin-4 receptors(IL-4R). IL-4-induced conditions include those arising from biologicalresponses that result from the binding of IL-4 to a native IL-4 receptoron a cell, or which may be inhibited or suppressed by preventing IL-4from binding to an IL-4 receptor. Conditions that may be treatedinclude, but are not limited to, medical disorders characterized byabnormal or excess expression of IL-4, or by an abnormal host responseto IL-4 production. Further examples are conditions in whichIL-4-induced antibody production, or proliferation or influx of aparticular cell type, plays a role. IL-4-induced disorders include thosein which IL-4 induces upregulation of IL-4 receptors or enhancedproduction of another protein that plays a role in a disease (e.g.,another cytokine).

A method for treating a mammal, including a human patient, who has sucha medical disorder comprises administering an IL-4 antagonist to themammal or otherwise contacting endogenous IL-4 with an antagonist, e.g.,in an ex vivo procedure. Conditions that may be treated in accordancewith the present invention include, but are not limited to,septic/reactive arthritis, dermatitis herpetiformis, urticaria(especially chronic idiopathic urticaria), ulcers, gastric inflammation,mucosal inflammation, ulcerative colitis, Crohn's Disease, inflammatorybowel disease, other disorders of the digestive system in which IL-4plays a role (e.g., IL-4-induced inflammation of part of thegastrointestinal tract), conditions in which IL-4-induced barrierdisruption plays a role (e.g., conditions characterized by decreasedepithelial barrier function in the lung or gastrointestinal tract),scleroderma, hypertrophic scarring, Whipple's Disease, benign prostatehyperplasia, IL-4-induced pulmonary conditions (including those listedbelow), allergic reactions to medication, Kawasaki disease, sickle celldisease or crisis, Churg-Strauss syndrome, Grave's disease,pre-eclampsia, Sjogren's syndrome, autoimmune lymphoproliferativesyndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmuneuveitis, tuberculosis, nephrosis, pemphigus vulgaris or bullouspemphigoid (autoimmune blistering diseases), and myasthenia gravis (anautoimmune muscular disease). IL-4 antagonists also find use asadjuvants to allergy immunotherapy and as vaccine adjuvants, especiallywhen directing the immune response toward a TH1 response would bebeneficial in treating or preventing the disease in question.

Septic/Reactive Arthritis

IL-4 antagonists may be employed in treating septic arthritis, whichalso is known as reactive arthritis or bacterial arthritis. Septicarthritis can be triggered by (result from, or develop subsequent to)infection with such microbes as Staphylococcus aureus, Chlamydiatrachomatis, Yersinia e.g., Y. enterocolitica, Salmonella, e.g., S.enteritidis, Shigella and Campylobacter. S. aureus has been reported tobe the major human pathogen in septic arthritis, responsible for themajority of cases.

IL-4 and IL-4-dependent Th2 responses play roles in promoting septicarthritis. IL-4 antagonist(s) are employed in accordance with theinvention, to inhibit IL-4 and also to suppress the Th2 response inpatients having septic arthritis or at risk for developing septicarthritis.

IL-4 increases bacterial burden and bacterial persistence in joints, byinhibiting clearance of the bacteria. IL-4 antagonists may be employedto assist in the clearance of bacteria associated with reactivearthritis, thereby reducing clinical manifestations such as swelling injoints. IL-4 antagonists may be administered to a human patientafflicted with septic arthritis, to reduce IL-4-mediated jointinflammation. In one approach, an antagonist is injected into a joint,e.g., into synovial fluid in the knee.

The use of IL-4 antagonists may benefit patients having (or at risk for)septic arthritis by suppressing a TH2 response and promoting a TH1response against the infection. TH2 cytokines may contribute tobacterial persistence in the joint, whereas a TH1 response plays a rolein eliminating the bacteria.

The antagonists may be administered to patients infected with bacteriaor other microbes such as those listed above, to prevent development ofseptic arthritis. Antagonist(s) may be administered after diagnosis withsuch an infection, but before development of clinical symptoms of septicarthritis.

Whipple's Disease

Tropheryma whippelii is the causative bacterium for Whipple's Disease,also known as intestinal lipodystrophy and lipophagia granulomatosis.The disease is characterized by steatorrhea, frequently generalizedlymphadenopathy, arthritis, fever, and cough. Also reported in Whipple'sDisease patients are an abundance of “foamy” macrophages in the jejunallamina propria, and lymph nodes containing periodic acid-schiff positiveparticles appearing bacilliform by electron microscopy (Steadman'sMedical Dictionary, 26^(th) Edition, Williams & Wilkins, Baltimore, Md.,1995).

The use of IL-4 antagonist(s) may benefit patients having (or at riskfor developing) Whipple's Disease, by restoring a normal balance betweenthe TH1 and TH2 components of the patient's immune response. Increasedproduction of IL-4 (a TH2-type cytokine) and decreased levels of certainTH1-type cytokines have been associated with Whipple's Disease. TH2cytokines may contribute to bacterial persistence, whereas a TH1response plays a role in clearing the causative bacteria. IL-4antagonists may be administered to patients infected with T. whippelii,whether or not the patient exhibits clinical symptoms of Whipple'sDisease.

Dermatitis Herpetiformis

Dermatitis herpetiformis, also known as Duhring's disease, is a chronicskin condition characterized by blistering skin lesions, cutaneous IgAdeposits, and itching. Patients have an immunobullous skin disorder withan associated gluten sensitive enteropathy, which is mediated by a Th2immune response. IL-4 antagonist(s) are administered in accordance withthe present invention, to inhibit IL-4 and the Th2 response, thuspromoting healing of current lesions and reducing or preventing theformation of blisters on the extensor body surfaces.

Hypertrophic Scarring

In accordance with the present invention, IL-4 antagonist(s) areadministered to patients who have, or are susceptible to developing,hypertrophic scarring. In one method provided herein, an IL-4 antagonistis administered to a burn patient. An immune response to burns and otherinjury is believed to play a role in the pathogenesis of hypertrophicscarring. Increased production of TH2-type cytokines, including IL-4,and reduced levels of certain TH1-type cytokines have been reported inburn patients who have hypertropic scarring. The use of IL-4 antagonistsmay benefit patients having (or at risk for developing) hypertrophicscarring, by suppressing a TH2-type immune response.

Urticaria

Urticaria, especially chronic forms thereof such as chronic idiopathicurticaria (CIU), may be treated with an IL-4 antagonist in accordancewith the present invention. CIU patients have higher serum levels ofIL-4 than controls, and may have a predominantly TH2-type cytokineprofile. Mast cells and Th2-type T cells are implicated as primaryeffector cells in chronic urticaria. IL-4 stimulates mast cellproliferation. Mast cell degranulation leads to histamine release,subsequent erythema, eosinophilia, redness of skin, and itching. IL-4antagonists are administered to inhibit IL-4 and reduce the TH2-typeresponse, thereby helping to control a patient's urticaria.

Ulcerative Colitis; Other Disorders of the Gastrointestinal Tract

IL-4 is implicated in the pathogenesis of ulcerative colitis. Th2-typecytokines including IL-4 may predominate in the colonic mucosa ofpatients with this disorder. The use of IL-4 antagonist(s) to suppressthe TH2 response may alleviate this condition.

In addition to ulcerative colitis, other disorders of thegastrointestinal tract or digestive system may be treated with IL-4antagonist(s). Examples of such disorders include, but are not limitedto, inflammatory bowel disease (IBD), with ulcerative colitis andCrohn's Disease being forms of IBD, gastritis, ulcers, and mucosalinflammation.

Any gastrointestinal condition in which IL-4 plays a role may be treatedwith an IL-4 antagonist in accordance with the present invention. Forexample, conditions involving IL-4-induced inflammation of part of thegastrointestinal tract may be treated with an IL-4 antagonist.Particular embodiments are directed to treatment of chronic inflammatoryconditions in the gastrointestinal tract.

Other embodiments are directed to conditions in which IL-4-inducedbarrier disruption plays a role, e.g., conditions characterized bydecreased epithelial barrier function in at least a portion of thegastrointestinal tract. Such conditions may, for example, involve damageto the epithelium that is induced by IL-4, directly or indirectly.

The intestinal epithelium forms a relatively impermeable barrier betweenthe lumen and the submucosa. Disruption of the epithelial barrier hasbeen associated with conditions such as inflammatory bowel disease. Seethe discussion in Youakim, A. and M. Ahdieh (Am. J. Physiol. 276(Gastrointest. Liver Physiol. 39):G1279-G1288, 1999), herebyincorporated by reference in its entirety. A damaged or “leaky” barriercan allow antigens to cross the barrier, which in turn elicits an immuneresponse that may cause further damage to gastrointestinal tissue. Suchan immune response may include recruitment of neutrophils or T cells,for example. An IL-4 antagonist may be administered to inhibitundesirable stimulation of an immune response.

Lung Disorders

Methods for treating IL-4-induced pulmonary disorders are providedherein. Such disorders include, but are not limited to, lung fibrosis,including chronic fibrotic lung disease, other conditions characterizedby IL-4-induced fibroblast proliferation or collagen accumulation in thelungs, pulmonary conditions in which a TH2-type immune response plays arole, conditions characterized by decreased barrier function in the lung(e.g., resulting from IL-4-induced damage to the epithelium), orconditions in which IL-4 plays a role in an inflammatory response.

Cystic fibrosis is characterized by the overproduction of mucus anddevelopment of chronic infections. Inhibiting IL-4 and the Th2 responsewill reduce mucus production and help control infections such asallergic bronchopulmonary aspergillosis (ABPA).

Allergic bronchopulmonary mycosis occurs primarily in patients withcystic fibrosis or asthma, where a Th2 immune response is dominant.Inhibiting IL-4 and the Th2 response will help clear and control theseinfections.

Chronic obstructive pulmonary disease is associated with mucushypersecrection and fibrosis. Inhibiting IL-4 and the Th2 response willreduce the production of mucus and the development of fibrous therebyimproving respiratory function and delaying disease progression.

Bleomycin-induced pneumopathy and fibrosis, and radiation-inducedpulmonary fibrosis are disorders characterized by fibrosis of the lungwhich is manifested by the influx of Th2, CD4 cells and macrophages,which produce IL-4 which in turn mediates the development of fibrosis.Inhibiting IL-4 and the Th2 response will reduce or prevent thedevelopment of these disorders.

Pulmonary alveolar proteinosis is characterized by the disruption ofsurfactant clearance. IL-4 increases surfactant product. Use of IL-4antagonists will decrease surfactant production and decrease the needfor whole lung lavage.

Adult respiratory distress syndrome (ARDS) may be attributable to anumber of factors, one of which is exposure to toxic chemicals. Onepatient population susceptible to ARDS is critically ill patients who goon ventilators. ARDS is a frequent complication in such patients. IL-4antagonists may alleviate ARDS by reducing inflammation and adhesionmolecules, although methods for treating such patients in accordancewith the present invention are not limited by a particular mechanism ofaction. IL-4 antagonists may be used to prevent or treat ARDS.

Sarcoidosis is characterized by granulomatus lesions. Use of IL-4antagonists to treat sarcoidosis, particularly pulmonary sarcoidosis, iscontemplated herein.

Conditions in which IL-4-induced barrier disruption plays a role (e.g.,conditions characterized by decreased epithelial barrier function in thelung) may be treated with IL-4 antagonist(s). Damage to the epithelialbarrier in the lungs may be induced by IL-4 directly or indirectly. Theepithelium in the lung functions as a selective barrier that preventscontents of the lung lumen from entering the submucosa. A damaged or“leaky” barrier allows antigens to cross the barrier, which in turnelicits an immune response that may cause further damage to lung tissue.Such an immune response may include recruitment of eosinophils or mastcells, for example. An IL-4 antagonist may be administered to inhibitsuch undesirable stimulation of an immune response.

IL-4 antagonists may be employed to promote healing of lung epithelium,thus restoring barrier function. IL-4 antagonists may be employed topromote healing of lung epithelium in asthmatics, for example.Alternatively, the antagonist is administered for prophylactic purposes,to prevent IL-4-induced damage to lung epithelium.

Tuberculosis

A TH2-type immune response is implicated in playing a role in causingtissue damage (e.g., necrosis of lung tissue) in tuberculosis (TB)patients. Elevated levels of IL-4 are associated with TB. IL-4production may be particularly elevated in cavitary tuberculosis (i.e.,in TB patients who have developed pulmonary cavities, which can bedetected/visualized by such techniques as radiographs of the chest).

IL-4 antagonists may benefit TB patients (especially those with cavitaryTB) by suppressing a TH2-type immune response, or by binding (andinactivating) excess secreted IL-4. Methods for treating such patientsin accordance with the present invention are not limited by a particularmechanism of action, however. IL-4 antagonists advantageously areadministered in an amount that restores the desired balance between theTH1 and TH2 components of the immune response, and reduces IL-4-inducedtissue damage in a patient.

Churg-Strauss Syndrome

Churg-Strauss syndrome, a disease also known as allergic granulomatousangiitis, is characterized by inflammation of the blood vessels inpersons with a history of asthma or allergy, and by eosinophilia. IL-4antagonist(s) may be administered to alleviate inflammation in patientswith this syndrome. The use of IL-4 antagonists to suppress a TH2-typeimmune response, and to combat eosinophilia, would benefit the patients.

Pre-Eclampsia

Pre-eclampsia is a toxemia of late pregnancy. The condition ischaracterized by a sharp rise in blood pressure, generally accompaniedby edema and albuminuria, during the third term of pregnancy.

Elevated TH1-type and TH2-type immune responses may play a role in thecondition. One method provided herein comprises administering an IL-4antagonist to a pregnant woman who has developed pre-eclampsia. The IL-4antagonist is administered in an amount, and for a period of time,sufficient to reduce the level of IL-4 (or of TH2-type cytokinescollectively) to a level that is considered normal during pregnancy. Ingeneral, the IL-4 antagonist is administered repeatedly throughout theduration of the pregnancy.

Scleroderma

IL-4 antagonist(s) are administered to scleroderma patients inaccordance with the invention. The antagonists reduce IL-4-inducedcollagen synthesis by fibroblasts in the patients. The antagonists maybe employed in preventing or reducing fibrosis in skin and lung tissues,as well as other tissues in which fibrosis occurs in sclerodermapatients, suppressing collagen synthesis in such tissues, and intreating scleroderma-related pulmonary disease.

Benign Prostate Hyperplasia

Benign prostate hyperplasia (BPH), also known as benign prostatehypertrophy, may be treated with IL-4 antagonist(s). While not wishingto be bound by a particular mechanism of action, administration of anIL-4 inhibitor may benefit a patient with BPH by suppressingIL-4-induced inflammation, or by suppressing a TH2-type immune response.

Grave's Disease

Antibodies directed against thyrotropin receptor play an important rolein Grave's Disease, a disorder characterized by hyperthyroidism. Studiesof cytokine production in Grave's Disease patients show a shift toward aTH2-type cytokine response. Use of an IL-4 antagonist to suppress theTH2-type immune response, and suppress antibody production, wouldbenefit Grave's Disease patients.

Sickle Cell Disease

Sickle cell disease patients typically experience intermittent periodsof acute exacerbation called crises, with the crises being categorizedas anemic or vaso-occlusive. IL-4 antagonists find use in treating orpreventing sickle cell crisis, especially in patients with elevated IL-4levels or in whom the immune response has shifted toward a TH2-typeresponse. Sickle cell disease (especially sickle cell crisis) has beenassociated with increased susceptibility to infectious diseases,including bacterial infections. Administering IL-4 antagonists to sicklecell disease patients may help the patient mount an immune responseagainst infectious diseases.

Sjogren's Syndrome

The autoimmune disease known as Sjogren's syndrome or sicca syndrometypically combines dry eyes and dry mouth with a disorder of theconnective tissues, such as rheumatoid arthritis, lupus, scleroderma, orpolymyositis. The vast majority of patients are middle age (or older)females. Sjogren's syndrome is an inflammatory disease of glands (e.g.,lacrimal and salivary glands) and other tissues of the body. Thesyndrome typically is associated with autoantibody production.

IL-4 antagonists may be administered to reduce the inflammatory response(such as inflammation of glands, including lacrimal glands) in suchpatients. IL-4 antagonists may benefit Sjogren's syndrome patients bysuppressing a TH2-type immune response, or by binding (and inactivating)excess IL-4 at inflammatory lesions. Methods for treating patients inaccordance with the present invention are not limited by a particularmechanism of action, however.

Autoimmune Lymphoproliferative Syndrome

Manifestations of autoimmune lymphoproliferative syndrome includelymphoproliferation and autoantibody production. Patients with thesyndrome reportedly have an inherited deficiency in apoptosis. IL-4antagonists may benefit patients with this syndrome by suppressing aTH2-type immune response, or by binding (and inactivating) excess IL-4at sites of inflammation. Methods for treating such patients inaccordance with the present invention are not limited by a particularmechanism of action, however.

Autoimmune Hemolytic Anemia

Excessive IL-4 secretion, and a deficiency in TH1-type cytokines, areimplicated in contributing to the pathogenesis of autoimmune hemolyticanemia. IL-4 antagonists are administered in accordance with the presentinvention, to benefit the patients by reducing autoantibody production,and by restoring a more normal balance between the TH1 and TH2components of the immune response.

Autoimmune Uveitis

Uveitis involves inflammation of the uvea (generally considered toinclude the iris, ciliary body, and choroid, considered together).Excess IL-4 secretion is implicated as playing a role in pathogenesis ofthis sight-threatening inflammatory eye disease. In accordance with thepresent invention, IL-4 antagonist(s) are administered to a uveitispatient to reduce disease severity. In one embodiment, IL-4antagonist(s) are administered to an individual who has autoimmuneuveoretinitis.

Kawasaki Disease

Also known as the mucocutaneous lymph node syndrome, Kawasaki disease(KD) mainly afflicts young children. The disease is characterized byparticular changes in the mucus membranes lining the lips and mouth, andby enlarged, tender lymph glands. Symptoms typically include fever,conjunctivitis, inflammation of the lips and mucous membranes of themouth, swollen glands in the neck, and a rash covering the hands andfeet, leading to hardened, swollen and peeling skin on hands and feet.In children with Kawasaki Disease (KD), inflammation of arteries(vasculitis) may develop. Due to the effect of the disease on thevascular system, KD reportedly is the main cause of acquired heartdisease in children.

IL-4 antagonists may be administered to patients with Kawasaki Disease,to reduce the elevated levels of IL-4 in the patient. Excessive IL-4secretion and a deficiency in TH1-type cytokines contribute to thepathogenesis of the disease.

Barrett's Esophagus

Barrett's esophagus is a condition characterized by alteration(subsequent to irritation) of the cells in the epithelial tissue thatlines the lower portion of the esophagus. Frequent reflux of the stomachcontents into the esophagus, over time, can lead to Barrett esophagus.Patients with Barrett esophagus are at risk for developing esophagealcancer (e.g., adenocarcinoma). While not wishing to be bound by aparticular mechanism of action, administration of an IL-4 antagonist maybenefit a patient with Barrett's esophagus by suppressing a TH2-typeimmune response. In one embodiment, an IL-4 antagonist is administeredto a patient with esophagitis, to inhibit progression to Barrett'sesophagus.

Nephrosis

Nephrosis, also known as nephrotic syndrome, is kidney disease that isnon-inflammatory and non-malignant. In the condition known as minimalchange nephrosis, glomerular damage (believed to arise from structuralchanges in glomerular visceral epithelial cells) results inabnormalities that include proteinuria. A TH2-type immune response(especially secretion of the TH2-type cytokines IL-4 and IL-13) areimplicated as playing a role in pathogenesis of minimal changenephrosis.

Other Indications

Additional examples of conditions that may be treated in accordance withthe present invention include but are not limited to the following. IL-4antagonists may be employed in treating or preventing hyper IgEsyndrome, idiopathic hypereosinophil syndrome, allergic reactions tomedication, autoimmune blistering diseases (e.g., pemphigus vulgaris orbullous pemphigoid), myasthenia gravis (an autoimmune muscular disease),and chronic fatigue syndrome. IL-4 inhibitors may be employed intreating GVHD; particular methods for treating GVHD combination therapywith other therapeutic agents as described below. IL-4 inhibitors alsofind use in treating or preventing hepatotoxicity induced by drugs suchas diclofenac (a non-steroidal anti-inflammatory drug).

An IL-4 antagonist may be employed as an adjuvant to allergyimmunotherapy treatment. IL-4 antagonists find further use as vaccineadjuvants, such as adjuvants for cancer vaccines and infectious diseasevaccines. The use of IL-4 antagonists is especially advantageous whenfavoring a TH1-type immune response would be beneficial in preventing ortreating the condition for which the vaccine is being administered. IL-4antagonists may be employed when reducing an antibody-mediated immuneresponse and/or promoting a T-cell-mediated immune response is desired.

IL-4 Antagonists

IL-4 antagonists that may be employed in accordance with the presentinvention include compounds that inhibit a biological activity of IL-4.The IL-4-induced biological activities to be inhibited by the methodsprovided herein are activities that directly or indirectly play a rolein the condition to be treated.

Examples of IL-4 antagonists include, but are not limited to, IL-4receptors (IL-4R), antibodies, other IL-4-binding molecules, and IL-4muteins as discussed further below. The antibodies may bind IL-4 or maybind an IL-4 receptor, for example.

Antagonists that bind IL-4 include but are not limited to IL-4 receptorsand anti-IL-4 antibodies. Endogenous IL-4 that becomes bound to such anantagonist is thereby prevented from binding its natural receptor oncell surfaces in vivo, and thus cannot manifest IL-4-mediated biologicalactivities.

Different types of antagonists may act at different sites or bydifferent mechanisms of action. Examples include but are not limited toantagonists that interfere with binding of IL-4 to cell surfacereceptors or that inhibit signal transduction. The site of action may beintracellular (e.g., by interfering with an intracellular signalingcascade), on a cell surface, or extracellular. Antagonists that act byinterfering with the interaction of IL-4 with IL-4R may bind to eitherIL-4 or to the receptor. An antagonist need not completely inhibit anIL-4 induced activity to find use in the present invention; rather,antagonists that reduce a particular activity of IL-4 are contemplatedfor use as well.

The above-presented discussions of particular mechanisms of action forIL-4 antagonists in treating particular diseases are illustrative only,and the methods presented herein are not bound thereby. The mechanismsof action by which IL-4 antagonists ameliorate diseases are not limitedto those discussed above.

In treating particular disorders, an IL-4 antagonist may reduce theamount of active IL-4 at a particular site within the body that isinvolved in the disorder. Antagonists that bind IL-4 such that it nolonger can bind to endogenous cellular receptors functionally reduce theamount of active IL-4 available for inducing biological responses.

An IL-4 antagonist may alleviate a disorder by reducing the amount offree endogenous IL-4 that is circulating in the body, e.g., in thebloodstream or in a particular tissue. When the action of IL-4 on suchtissue plays a role in pathogenesis of the disease, the antagonistserves to block action of IL-4 in the tissue, thereby alleviating thedisorder. In a further example, antagonists may inhibit IL-4-inducedrecruitment of cells to a site or tissue within the body, wherein suchrecruitment plays a role in causing or exacerbating a disease. Theantagonists may inhibit an IL-4-mediated influx of cells involved in animmune or inflammatory response. An antagonist may act by reducingproliferation, activation, migration, influx, or accumulation of aparticular cell type, or by inhibiting a biological response directly orindirectly attributable to a particular cell type. Examples ofparticular cell types are fibroblasts, mast cells, and eosinophils.

As discussed above, some conditions may be treated by suppressing aTH2-type immune response. IL-4 is associated with a TH2 response, and isone of the cytokines secreted by T-helper cells of type 2 (TH2 cells).An IL-4 antagonist may be administered to reduce a TH2-type immuneresponse. The IL-4 antagonist may be said to reduce proliferation of TH2cells, to suppress a TH2 response, to shift the immune response toward aTH1 response, or to favor a TH1-type response. The use of antagonists ofother cytokines associated with a TH2-type immune response is discussedbelow. Antagonists of other TH2-type cytokine(s), such as IL-5, IL-10,or IL-13, may be administered to patients who have a disorder involvingelevated levels of such cytokines. Techniques for measuring the amountof such cytokines in a patient, e.g., in the patient's serum, are wellknown.

One embodiment of the invention is directed to a method for inhibitingIL-4-induced damage to epithelium, comprising administering an IL-4antagonist to an individual who has, or is at risk of developing, acondition in which IL-4-mediated epithelial barrier disruption plays arole. In accordance with the present invention, barrier function studiesrevealed that IL-4 plays a role in reduction of barrier function inmodels of lung epithelium and intestinal epithelium, and that a solublehuman IL-4 receptor polypeptide (an IL-4 antagonist) inhibits theIL-4-mediated reduction of barrier function (see example 7).

Particular embodiments of methods provided herein comprise administeringan IL-4 antagonist to inhibit IL-4-induced damage to epithelium in thegastrointestinal tract or lung. Such methods may be employed to preventepithelial damage, or to restore epithelial barrier function (i.e.,promote repair or healing of the epithelium). The ability of an IL-4antagonist to inhibit IL-4-induced damage to epithelium may be confirmedin any of a number of suitable assays, such as those described inexample 7 below.

Any inflammation associated with (or subsequent to) an infection alsomay be treated with an IL-4 antagonist. The antagonist may beadministered to inhibit any IL-4-induced component of an inflammatoryresponse resulting from microbial infection in the gastrointestinaltract, for example.

Combinations of two or more antagonists may be employed in methods andcompositions of the present invention. Examples of suitable IL-4antagonists are as follows.

IL-4 Receptor

A preferred IL-4 antagonist is an IL-4 receptor (IL-4R). Whenadministered in vivo, IL-4R polypeptides circulate in the body and bindto circulating endogenous IL-4 molecules, preventing interaction of IL-4with endogenous cell surface IL-4 receptors, thus inhibitingtransduction of IL-4-induced biological signals.

IL-4 receptors are described in U.S. Pat. No. 5,599,905; Idzerda et al.,J. Exp. Med. 171:861-873, March 1990 (human IL-4R); and Mosley et al.,Cell 59:335-348, Oct. 20, 1989 (murine IL-4R); each of which is herebyincorporated by reference. The protein described in those threereferences is sometimes referred to in the scientific literature asIL-4Rα. Unless otherwise specified, the terms “IL-4R” and “IL-4receptor” as used herein encompass this protein in various forms thatare capable of functioning as IL-4 antagonists, including but notlimited to soluble fragments, fusion proteins, oligomers, and variantsthat are capable of binding IL-4, as described in more detail below.

The nucleotide sequence of a human IL-4R cDNA, and the amino acidsequence encoded thereby, are set forth in FIGS. 1A-1C. The cDNA clonewas isolated from a cDNA library derived from a CD4⁺/CD8⁻ human T cellclone designated T22, as described in Idzerda et al., J. Exp. Med.,171:861, March 1990, and in U.S. Pat. No. 5,599,905, which are herebyincorporated by reference in their entirety. The DNA and amino acidsequences of FIGS. 1A-1C are presented in SEQ ID NO:1 and SEQ ID NO:2,respectively.

The encoded human IL-4R protein comprises (from N- to C-terminus) anN-terminal signal peptide, followed by an extracellular domain, atransmembrane region, and a cytoplasmic domain. The transmembraneregion, which is underlined in FIG. 1A, corresponds to amino acids 208through 231. The cytoplasmic domain comprises amino acids 232 through800.

A signal peptide includes amino acids −25 to −1 of SEQ ID NO:2. Analternative signal peptide cleavage site occurs between residues −3 and−2 of SEQ ID NO:2, such that the signal peptide corresponds to residues−25 through −3.

As is recognized in the pertinent field, the signal peptide cleavagesite for a given protein may vary according to such factors as theparticular expression system (especially the host cells) in which theprotein is expressed. The exact boundaries of the signal peptide, andthus the extracellular domain, of a given recombinant protein thus maydepend on the expression system employed. Further, the signal peptidemay be cleaved at more than one position, generating more than onespecies of polypeptide in a preparation of recombinant protein.

In one embodiment, in which an expression vector comprises DNA encodingamino acids −25 through 207 of SEQ ID NO:2, the expressed recombinantIL-4R includes two species of mature soluble human IL-4R. The expressedpolypeptides include a major species corresponding to amino acids −2 to207 and a minor species corresponding to amino acids 1 to 207 of SEQ IDNO:2. Two alternate forms of the extracellular domain of human IL-4Rthus correspond to residues −2 to 207 and 1 to 207 of SEQ ID NO:2. Theterm “mature” refers to a protein in a form lacking a signal peptide orleader sequence, as is understood in the pertinent art.

Among the IL-4 receptors suitable for use herein are IL-4R fragments.Truncated IL-4R polypeptides may occur naturally, e.g., as a result ofproteolytic cleavage, post-translational processing, or alternativesplicing of mRNA. Alternatively, fragments may be constructed bydeleting terminal or internal portions of an IL-4R sequence, e.g., viarecombinant DNA technology. Fragments that retain the ability to bindIL-4 may be identified in conventional binding assays. Such fragmentsmay be soluble fragments, as discussed below.

In a preferred embodiment of the invention, the antagonist comprises asoluble form of the IL-4R. A soluble IL-4 receptor is a polypeptide thatis secreted from the cell in which it is expressed, rather than beingretained on the cell surface. The full length human IL-4R protein of SEQID NO:2 is a transmembrane protein, which, as described above, comprisesan N-terminal signal peptide, followed by an extracellular domain, atransmembrane region, and a C-terminal cytoplasmic domain. Soluble IL-4Rpolypeptides lack the transmembrane region that would cause retention onthe cell, and the soluble polypeptides consequently are secreted intothe culture medium. The transmembrane region and intracellular domain ofIL-4R may be deleted or substituted with hydrophilic residues tofacilitate secretion of the receptor into the cell culture medium.

Particular embodiments of soluble IL-4R polypeptides lack thetransmembrane region but comprise the extracellular domain (the completeextracellular domain or a fragment thereof that is capable of bindingIL-4). As one option, the polypeptide comprises all or part of thecytoplasmic domain, as well as the extracellular domain (or fragment ofthe extracellular domain), but lacks the transmembrane region.

Examples of soluble human IL-4R polypeptides include, but are notlimited to, polypeptides comprising amino acid residues x to y of SEQ IDNO:2, wherein x represents 1 or −2 and y represents an integer from 197to 207. Preferred embodiments include polypeptides comprising residues 1to 207 or −2 to 207 of SEQ ID NO:2.

A protein preparation administered as an IL-4 antagonist may comprisemore than one form of IL-4R. For example, the preparation may comprisepolypeptide molecules consisting of amino acids 1 to 207 of SEQ ID NO:2,as well as polypeptides consisting of amino acids −2 to 207 of SEQ IDNO:2.

IL-4R polypeptides arising from alternative mRNA constructs, e.g., whichcan be attributed to different mRNA splicing events followingtranscription, and which yield polypeptide translates capable of bindingIL-4, are among the IL-4R polypeptides disclosed herein. Suchalternatively spliced mRNAs may give rise to soluble polypeptides.

Further examples of IL-4 receptors that may be employed in the methodsprovided herein are variants having amino acid sequences which aresubstantially similar to the native interleukin-4 receptor amino acidsequence of SEQ ID NO:2, or fragments thereof. Variant IL-4 receptorpolypeptides that are capable of functioning as IL-4 antagonists may beemployed in the methods of the present invention.

Any of a number of conventional assay techniques may be employed toconfirm that a given form of IL-4R (e.g., an IL-4R fragment or variant)functions as an IL-4 antagonist. Examples include binding assays orassays that test the ability of a given IL-4R polypeptide to inhibittransduction of an IL-4-induced biological signal. Examples of suitablein vitro assays are described below.

“Substantially similar” IL-4 receptors include those having amino acidor nucleic acid sequences that vary from a native sequence by one ormore substitutions, deletions, or additions, but retain a desiredbiological activity of the IL-4R protein. Examples of nucleic acidmolecules encoding IL-4 receptors include, but are not limited to: (a)DNA derived from the coding region of a native mammalian IL-4R gene; (b)DNA that is capable of hybridization to a DNA of (a) under moderatelystringent conditions and which encodes an IL-4R having a biologicalactivity of a native IL-4R; or (c) DNA that is degenerate as a result ofthe genetic code to a DNA defined in (a) or (b) and which encodes anIL-4R having a biological activity of a native IL-4R. Due to codedegeneracy, there can be considerable variation in nucleotide sequencesencoding the same amino acid sequence.

Variants may be naturally occurring, such as allelic variants or thosearising from alternative splicing of mRNA. Alternatively, variants maybe prepared by such well known techniques as in vitro mutagenesis.

A variant sequence identified by Idzerda et al., supra, comprises a GTCcodon encoding the amino acid valine (Val) at position 50, instead ofisoleucine (lile). The variant sequence is otherwise identical to thesequence of SEQ ID NOS:1 and 2. IL-4R fragments, such as solublefragments, comprising Val at position 50 are provided herein.

In particular embodiments, an IL-4 receptor DNA or amino acid sequenceis at least 80 percent identical to the sequence of a native IL-4R.Preferably, an IL-4R DNA or polypeptide comprises a sequence that is atleast 90 percent identical to a native IL-4R DNA or amino acid sequence.One example is a human IL-4R comprising an amino acid sequence that isat least 80 percent identical to the sequence presented in SEQ ID NO:2.Another example is a soluble IL-4R comprising an amino acid sequence atleast 80 percent identical to the sequence of the extracellular domainof human IL-4R. Further examples are polypeptides comprising amino acidsequences that are at least 90 percent identical to the sequencepresented in SEQ ID NO:2, or a fragment thereof. In a particularembodiment, the polypeptide comprises no more than 10 amino acidsubstitutions. IL-4R polypeptides that retain the ability to bind IL-4may be identified in conventional binding assays.

Percent similarity or percent identity may be determined, for example,by comparing DNA or amino acid sequence information using the GAPcomputer program, version 6.0, available from the University ofWisconsin Genetics Computer Group (UWGCG). The GAP program utilizes thealignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970),as revised by Smith and Waterman (Adv. Appl. Math. 2:482, 1981).Briefly, the GAP program defines similarity as the number of alignedsymbols (i.e., nucleotides or amino acids) which are similar, divided bythe total number of symbols in the shorter of the two sequences. Thepreferred default parameters for the GAP program include: (1) a unarycomparison matrix (containing a value of 1 for identities and 0 fornon-identities) for nucleotides, and the weighted comparison matrix ofGribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described bySchwartz and Dayhoff, ed., Atlas of Protein Sequence and Structure,National Biomedical Research Foundation, pp. 353-358, 1979; (2) apenalty of 3.0 for each gap and an additional 0.10 penalty for eachsymbol in each gap; and (3) no penalty for end gaps.

IL-4R polypeptides that vary from native proteins but possess a desiredproperty may be constructed by, for example, substituting or deletingresidues not needed for the particular biological activity.Substitutions may be conservative substitutions, such that a desiredbiological property of the protein is retained. Amino acids may bereplaced with residues having similar physicochemical characteristics.

Cysteine residues can be deleted or replaced with other amino acids toprevent formation of incorrect intramolecular disulfide bridges uponrenaturation. Other alterations of a native sequence involvemodification of adjacent dibasic amino acid residues, to enhanceexpression in yeast host cells in which KEX2 protease activity ispresent.

The present invention also includes IL-4R with or without associatednative-pattern glycosylation. The glycosylation pattern may varyaccording to the type of host cells in which the protein is produced.Another option is inactivation of N-glycosylation sites by site-specificmutagenesis. N-glycosylation sites in eukaryotic proteins arecharacterized by the amino acid triplet Asn-A₁-Z, where A₁ is any aminoacid except Pro, and Z is Ser or Thr. In this sequence, asparagineprovides a side chain amino group for covalent attachment ofcarbohydrate. Such a site can be eliminated by substituting anotheramino acid for Asn or for residue Z, deleting Asn or Z, or inserting anon-Z amino acid between A₁ and Z, or an amino acid other than Asnbetween Asn and A₁.

Oligonucleotide-directed site-specific mutagenesis procedures can beemployed to provide an altered gene having particular codons alteredaccording to the substitution, deletion, or insertion required. Examplesof techniques for making such alterations are described in Walder et al.(Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik(BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering:Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos.4,518,584 and 4,737,462.

IL-4 receptors that may be employed also include derivatives, e.g.,various structural forms of the primary protein which retain a desiredbiological activity. Due to the presence of ionizable amino and carboxylgroups, for example, an IL-4R protein may be in the form of acidic orbasic salts, or in neutral form. Individual amino acid residues may alsobe modified by oxidation or reduction. The primary amino acid structuremay be modified by forming covalent or aggregative conjugates with otherchemical moieties, such as glycosyl groups, lipids, phosphate, acetylgroups and the like, or by creating amino acid sequence mutants.PEGylated derivatives (modified with polyethylene glycol) arecontemplated. Covalent derivatives may be prepared by linking particularfunctional groups to IL-4R amino acid side chains or at the N- orC-termini. IL-4R derivatives may also be obtained by cross-linkingagents, such as M-maleimidobenzoyl succinimide ester andN-hydroxysuccinimide, at cysteine and lysine residues. IL-4R proteinsmay also be covalently bound through reactive side groups to variousinsoluble substrates, such as cyanogen bromide-activated,bisoxirane-activated, carbonyldiimidazole-activated or tosyl-activatedagarose structures, or by adsorbing to polyolefin surfaces (with orwithout glutaraldehyde cross-linking).

Other derivatives of IL-4R within the scope of this invention includecovalent or aggregative conjugates of IL-4R or its fragments with otherproteins or polypeptides, such as by expression of recombinant fusionproteins comprising heterologous polypeptides fused to the N-terminus orC-terminus of an IL-4R polypeptide. For example, the conjugated peptidemay be a heterologous signal (or leader) polypeptide, e.g., the yeastα-factor leader, or a peptide such as an epitope tag. IL-4R-containingfusion proteins can comprise peptides added to facilitate purificationor identification of IL-4R (e.g., poly-His). Specific examples ofpoly-His fusion constructs that is biologically active are soluble humanIL-4R (e.g., comprising residues −2 to 207 or 1-207 of SEQ ID NO:2) HisHis and soluble human IL-4R His His His His His His. An amino acidsequence of IL-4 receptor can also be linked to the Flag® peptideAsp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (SEQ ID NO:3) as described inHopp et al., Bio/Technology 6:1204, 1988, and U.S. Pat. No. 5,011,912.The Flag® peptide is highly antigenic and provides an epitope reversiblybound by a specific monoclonal antibody, enabling rapid assay and facilepurification of expressed recombinant protein. Reagents useful forpreparing fusion proteins in which the Flag® peptide is fused to a givenpolypeptide are commercially available (Sigma, St. Louis, Mo.).

Oligomers that contain IL-4R polypeptides may be employed as IL-4antagonists. Oligomers may be in the form of covalently-linked ornon-covalently-linked dimers, trimers, or higher oligomers. Oligomerscomprising two or more IL-4R polypeptides are contemplated for use, withone example being a homodimer. Other oligomers include heterodimers,heterotrimers, and the like, which comprise an IL-4R polypeptide as wellas at least one polypeptide that is not derived from the IL-4R of SEQ IDNO:2.

One embodiment is directed to oligomers comprising multiple IL-4Rpolypeptides joined via covalent or non-covalent interactions betweenpeptide moieties fused to the IL-4R polypeptides. Such peptides may bepeptide linkers (spacers), or peptides that have the property ofpromoting oligomerization. Leucine zippers and certain polypeptidesderived from antibodies are among the peptides that can promoteoligomerization of IL-4R polypeptides attached thereto, as described inmore detail below.

In particular embodiments, the oligomers comprise from two to four IL-4Rpolypeptides. The IL-4R moieties of the oligomer may be in any of theforms described above, e.g., variants or fragments. Preferably, theoligomers comprise soluble IL-4R polypeptides.

As one alternative, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of fusion proteins comprising certainheterologous polypeptides fused to various portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al. (PNAS USA 88:10535, 1991); Byrn et al. (Nature 344:677,1990); and Hollenbaugh and Aruffo (“Construction of ImmunoglobulinFusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages10.19.1-10.19.11, 1992).

One embodiment of the present invention is directed to a dimercomprising two fusion proteins created by fusing IL-4R to the Fc regionof an antibody. A gene fusion encoding the IL-4R/Fc fusion protein isinserted into an appropriate expression vector. IL-4R/Fc fusion proteinsare expressed in host cells transformed with the recombinant expressionvector, and allowed to assemble much like antibody molecules, whereuponinterchain disulfide bonds form between the Fc moieties to yielddivalent IL-4R.

The term “Fc polypeptide” as used herein includes native and muteinforms of polypeptides derived from the Fc region of an antibody.Truncated forms of such polypeptides containing the hinge region thatpromotes dimerization are also included. Fusion proteins comprising Fcmoieties (and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

One suitable Fc polypeptide, described in PCT application WO 93/10151(hereby incorporated by reference), is a single chain polypeptideextending from the N-terminal hinge region to the native C-terminus ofthe Fc region of a human IgG1 antibody. Another useful Fc polypeptide isthe Fc mutein described in U.S. Pat. No. 5,457,035 and in Baum et al.,(EMBO J. 13:3992-4001, 1994). The amino acid sequence of this mutein isidentical to that of the native Fc sequence presented in WO 93/10151,except that amino acid 19 has been changed from Leu to Ala, amino acid20 has been changed from Leu to Glu, and amino acid 22 has been changedfrom Gly to Ala. The mutein exhibits reduced affinity for Fc receptors.

In other embodiments, IL-4R may be substituted for the variable portionof an antibody heavy or light chain. If fusion proteins are made withboth heavy and light chains of an antibody, it is possible to form anoligomer with as many as four IL-4R extracellular regions.

Soluble recombinant fusion proteins comprising an IL-4R and variousportions of the constant region of an immunoglobulin are described in EP464,533, along with procedures for preparing such fusion proteins anddimers thereof. Among the fusion proteins described in EP 464,533 arethose comprising the extracellular portion of human IL-4R and an Fcpolypeptide.

Alternatively, the oligomer is a fusion protein comprising multipleIL-4R polypeptides, with or without peptide linkers (spacer peptides).Among the suitable peptide linkers are those described in U.S. Pat. Nos.4,751,180 and 4,935,233.

Another method for preparing oligomeric IL-4R involves use of a leucinezipper. Leucine zipper domains are peptides that promote oligomerizationof the proteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, 1988), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble oligomericproteins are described in PCT application WO 94/10308, and the leucinezipper derived from lung surfactant protein D (SPD) described in Hoppeet al. (FEBS Letters 344:191, 1994), hereby incorporated by reference.The use of a modified leucine zipper that allows for stabletrimerization of a heterologous protein fused thereto is described inFanslow et al. (Semin. Immunol. 6:267-278, 1994). In one approach,recombinant fusion proteins comprising a soluble IL-4R polypeptide fusedto a leucine zipper peptide are expressed in suitable host cells, andthe soluble oligomeric IL-4R that forms is recovered from the culturesupernatant.

One example of a heterodimer comprises an IL-4R polypeptide derived fromthe human IL-4R of SEQ ID NO:2, and an IL-2Rγ polypeptide. IL-2Rγ (alsoknown as IL-2Rγ_(c)) is described in U.S. Pat. No. 5,510,259 and inTakeshita et al. (Science 257:379, 17 Jul. 1992), which are incorporatedby reference herein. The polypeptides may be in one of the various formsdescribed herein, e.g., soluble fragments, variants, and the like,derived from the indicated proteins. One embodiment of such aheterodimer comprises a soluble IL-4R/Fc fusion protein and a solubleIL-2Rγ/Fc fusion protein. Such heterodimers are described in WO96/11213, along with IL-4R homodimers.

Other examples of heterodimers comprise an IL-4R subunit (preferably asoluble fragment of the protein of SEQ ID NO:2) and at least one IL-13receptor subunit. IL-13 receptor (IL-13R) complexes and IL-13Rpolypeptides (such as polypeptides designated IL-13Rα1 and IL-13Rα2) aredescribed in Zurawski et al., J. Biol. Chem. 270 (23), 13869, 1995; deVries, J. Allergy Clin. Immunol. 102(2):165, August 1998; Callard et al.Immunology Today, 17(3):108, March 1996, and U.S. Pat. No. 5,710,023,each of which is incorporated by reference herein. In one embodiment, aheterodimer comprises a soluble human IL-4R and a soluble IL-13R(preferably a soluble form of the polypeptide described in U.S. Pat. No.5,710,023 or IL-13Rα1). The components of heterodimers may be anysuitable form of the polypeptides that retains the desired activity,such as fragments, variants, or fusion proteins (e.g., fusions ofsoluble receptor polypeptides with Fc polypeptides, leucine zipperpeptides, peptide linkers, or epitope tags).

IL-4 receptor polypeptides and fusion proteins described herein may beprepared by any of a number of conventional techniques. IL-4Rpolypeptides may be purified from cells that naturally express thereceptor (such as the cells discussed in Park et al., Proc. Natl. Acad.Sci. USA 84:1669-673, 1987), or may be produced in recombinantexpression systems, using well known techniques. Expression systems foruse in producing IL-4R include those described in U.S. Pat. No.5,599,905, which is hereby incorporated by reference.

A variety of expression systems are known for use in producingrecombinant proteins. In general, host cells are transformed with arecombinant expression vector that comprises DNA encoding a desiredIL-4R polypeptide. Among the host cells that may be employed areprokaryotes, yeast or higher eukaryotic cells. Prokaryotes include gramnegative or gram positive organisms, for example E. coli or bacilli.Higher eukaryotic cells include insect cells and established cell linesof mammalian origin. Examples of suitable mammalian host cell linesinclude the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzmanet al., Cell 23:175, 1981), L cells, 293 cells, C127 cells, 3T3 cells(ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCCCRL 10) cell lines, and the CVI/EBNA cell line derived from the Africangreen monkey kidney cell line CVI (ATCC CCL 70) as described by McMahanet al. (EMBO J. 10: 2821, 1991). Appropriate cloning and expressionvectors for use with bacterial, fungal, yeast, and mammalian cellularhosts are described by Pouwels et al. (Cloning Vectors: A LaboratoryManual, Elsevier, New York, 1985).

The transformed cells are cultured under conditions that promoteexpression of the IL-4R, and the polypeptide is recovered byconventional protein purification procedures. One such purificationprocedure includes the use of affinity chromatography, e.g., over amatrix having IL-4 bound thereto. Expressed IL-4R will be deposited inthe cell membrane or secreted into the culture supernatant, depending onthe IL-4R DNA selected. Polypeptides contemplated for use herein includesubstantially homogeneous recombinant mammalian IL-4R polypeptidessubstantially free of contaminating endogenous materials.

Antibodies

Antibodies that function as IL-4 antagonists may be employed in themethods of the present invention. The antibodies preferably aremonoclonal antibodies or antigen-binding fragments thereof.Advantageously, humanized or chimeric monoclonal antibodies areemployed. Most preferred are human monoclonal antibodies prepared usingtransgenic mice, as described below.

Examples of suitable antibodies are those that interfere with thebinding of IL-4 to an IL-4 receptor. Such antibodies, referred to hereinas blocking antibodies, may be raised against either IL-4 or IL-4R, andscreened in conventional assays for the ability to interfere withbinding of IL-4 to IL-4 receptors. Examples of suitable assays areassays that test the antibodies for the ability to inhibit binding ofIL-4 to cells expressing IL-4R, or that test antibodies for the abilityto reduce a biological or cellular response that results from thebinding of IL-4 to cell surface IL-4 receptors.

It has been reported that IL-4Rα is a component of certain multi-subunitIL-13 receptor complexes (Zurawski et al., J. Biol. Chem. 270 (23),13869, 1995; de Vries, J. Allergy Clin. Immunol. 102(2):165, August1998; and Callard et al. Immunology Today, 17(3):108, March 1996, eachincorporated by reference herein). Thus, some antibodies raised againstIL-4Rα may interfere with the binding of IL-13 to such receptorcomplexes.

In one embodiment, antibodies directed against IL-4R block binding ofIL-4 and also IL-13 to cells. The antibodies inhibit IL-4-inducedbiological activity and also inhibit IL-13-induced activity, and thusmay be employed in treating conditions induced by either or bothcytokines. Examples of such conditions include but are not limited toIgE-mediated conditions, asthma, allergic conditions, allergic rhinitis,and dermatitis including atopic dermatitis.

Antibodies that bind to IL-4R and inhibit IL-4 binding may be screenedin various conventional assays to identify those antibodies that alsointerfere with the binding of IL-13 to such receptor complexes.Antibodies may be screened in binding assays or tested for the abilityto inhibit an IL-4-induced and an IL-13-induced biological activity. Anexample of a suitable assay is illustrated in Example 5 below.

Antibodies specific for IL-4 or IL-4R may be prepared by well knownprocedures. See, for example, Monoclonal Antibodies, Hybridomas: A NewDimension in Biological Analyses, Kennet et al. (eds.), Plenum Press,New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land(eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1988).

Antigen-binding fragments of such antibodies may be produced byconventional techniques. Examples of such fragments include, but are notlimited to, Fab and F(ab′)₂ fragments. Antibody fragments andderivatives produced by genetic engineering techniques are alsocontemplated for use. Unless otherwise specified, the terms “antibody”and “monoclonal antibody” as used herein encompass both whole antibodiesand antigen-binding fragments thereof.

Additional embodiments include chimeric antibodies, e.g., humanizedversions of murine monoclonal antibodies. Such humanized antibodies maybe prepared by known techniques, and offer the advantage of reducedimmunogenicity when the antibodies are administered to humans. In oneembodiment, a humanized monoclonal antibody comprises the variableregion of a murine antibody (or just the antigen binding site thereof)and a constant region derived from a human antibody. Alternatively, ahumanized antibody fragment may comprise the antigen binding site of amurine monoclonal antibody and a variable region fragment (lacking theantigen-binding site) derived from a human antibody. Procedures for theproduction of chimeric and further engineered monoclonal antibodiesinclude those described in Riechmann et al. (Nature 332:323, 1988), Liuet al. (PNAS 84:3439, 1987), Larrick et al. (Bio/Technology 7:934,1989), and Winter and Harris (TIPS 14:139, May, 1993).

A method for producing an antibody comprises immunizing a non-humananimal, such as a transgenic mouse, with an IL-4R polypeptide, wherebyantibodies directed against the IL-4R polypeptide are generated in saidanimal. Procedures have been developed for generating human antibodiesin non-human animals. The antibodies may be partially human, orpreferably completely human. For example, transgenic mice into whichgenetic material encoding one or more human immunoglobulin chains hasbeen introduced may be employed. Such mice may be genetically altered ina variety of ways. The genetic manipulation may result in humanimmunoglobulin polypeptide chains replacing endogenous immunoglobulinchains in at least some (preferably virtually all) antibodies producedby the animal upon immunization.

Mice in which one or more endogenous immunoglobulin genes have beeninactivated by various means have been prepared. Human immunoglobulingenes have been introduced into the mice to replace the inactivatedmouse genes. Antibodies produced in the animals incorporate humanimmunoglobulin polypeptide chains encoded by the human genetic materialintroduced into the animal.

Examples of techniques for production and use of such transgenic animalsare described in U.S. Pat. Nos. 5,814,318, 5,569,825, and 5,545,806,which are incorporated by reference herein. Examples 2-4 below providefurther description of the preparation of transgenic mice useful forgenerating human antibodies directed against an antigen of interest.

Antibodies produced by immunizing transgenic non-human animals with anIL-4R polypeptide are provided herein. Transgenic mice into whichgenetic material encoding human immunoglobulin polypeptide chain(s) hasbeen introduced are among the suitable transgenic animals. Examples ofsuch mice include, but are not limited to, those containing the geneticalterations described in the examples below. One example of a suitableimmunogen is a soluble human IL-4R, such as a polypeptide comprising theextracellular domain of the protein of SEQ ID NO:2, or other immunogenicfragment of the protein of SEQ ID NO:2.

Monoclonal antibodies may be produced by conventional procedures, e.g.,by immortalizing spleen cells harvested from the transgenic animal aftercompletion of the immunization schedule. The spleen cells may be fusedwith myeloma cells to produce hybridomas, by conventional procedures.

A method for producing a hybridoma cell line comprises immunizing such atransgenic animal with an IL-4R immunogen; harvesting spleen cells fromthe immunized animal; fusing the harvested spleen cells to a myelomacell line, thereby generating hybridoma cells; and identifying ahybridoma cell line that produces a monoclonal antibody that binds anIL-4R polypeptide. Such hybridoma cell lines, and anti-IL-4R monoclonalantibodies produced therefrom, are encompassed by the present invention.Monoclonal antibodies secreted by the hybridoma cell line are purifiedby conventional techniques. Hybridomas or MAbs may be further screenedto identify MAbs with particular properties, such as the ability toblock an IL-4-induced activity, and to block an IL-13-induced activity(see the assay in example 5).

Human antibodies that bind IL-4R are provided by the present invention.In one embodiment of the invention, human antibodies raised againstIL-4R and produced by techniques involving use of transgenic mice, blockbinding of IL-4 and also IL-13 to cells. Such antibodies are IL-4antagonists and additionally function as IL-13 antagonists.

Among the uses of antibodies directed against an IL-4R is use in assaysto detect the presence of IL-4R polypeptides, either in vitro or invivo. The antibodies also may be employed in purifying IL-4R proteins byimmunoaffinity chromatography. Those antibodies that additionally canblock binding of IL-4 to IL-4R may be used to inhibit a biologicalactivity that results from such binding. Blocking antibodies find use inthe methods of the present invention. Such antibodies which function asIL-4 antagonists may be employed in treating any IL-4-induced condition,including but not limited to asthma and allergies, e.g., allergicrhinitis, contact dermatitis, and atopic dermatitis. In one embodiment,a human anti-IL-4R monoclonal antibody generated by procedures involvingimmunization of transgenic mice is employed in treating such conditions.

Antibodies may be employed in an in vitro procedure, or administered invivo to inhibit an IL-4-induced biological activity. Disorders caused orexacerbated (directly or indirectly) by the interaction of IL-4 withcell surface IL-4 receptors thus may be treated. A therapeutic methodinvolves in vivo administration of a blocking antibody to a mammal in anamount effective for reducing an IL-4-induced biological activity.

Antibodies of the invention include, but are not limited to, partiallyhuman (preferably fully human) monoclonal antibodies that inhibit abiological activity of IL-4 and also inhibit a biological activity ofIL-13. One embodiment is directed to a human monoclonal antibody that atleast partially blocks binding of IL-4 to a cell, and at least partiallyblocks binding of IL-13 to a cell.

Antibodies of the present invention include but are not limited toantibodies generated by immunizing a transgenic mouse with an IL-4receptor immunogen, wherein the transgenic mouse is selected from themouse strains described in example 3 below. The desired antibodies areat least partially human, and preferably fully human. In one embodiment,the immunogen is a human IL-4 receptor polypeptide. Hybridoma cell linesderived from the thus-immunized mice, wherein the hybridoma secretes amonoclonal antibody that binds IL-4R, also are provided herein. Examplesof antibodies produced by immunizing such transgenic mice are the humanmonoclonal antibodies designated 6-2 (described in example 6); 12B5(described in example 8); and MAbs 63, 1B7, 5A1, and 27A1 (all describedin example 9). Monoclonal antibodies 6-2, 12B5, 63, 1B7, 5A1, and 27A1are fully human antibodies, and are capable of inhibiting activity ofboth IL-4 and IL-13. MAbs 12B5, 63, and 1B7 are preferred antagonists ofhuman IL-4 and human IL-13.

Particular monoclonal antibodies of the invention are selected from thegroup consisting of MAb 6-2; a Mab that is cross-reactive with 6-2; aMAb that binds to the same epitope as 6-2; a MAb that competes with 6-2for binding to a cell that expresses human IL-4R; a MAb that possesses abiological activity of 6-2; and an antigen-binding fragment of any ofthe foregoing antibodies. In one embodiment, the antibody has a bindingaffinity for human IL-4R that is substantially equivalent to the bindingaffinity of 6-2 for human IL-4R. MAb 6-2 is an IgM antibody. MAbs ofother isotypes (including but not limited to IgG1 and IgG4), derivedfrom 6-2, also are encompassed by the present invention. Hybridoma celllines that produce any such monoclonal antibodies also are provided bythe present invention.

One example of a biological activity of 6-2 is the ability to functionas both an IL-4 antagonist and an IL-13 antagonist. In one embodiment, aMAb of the invention possesses IL-4-blocking activity substantiallyequivalent to that of 6-2; and possesses IL-13-blocking activitysubstantially equivalent to that of 6-2. Such activity may be measuredin any suitable conventional assay (e.g., as measured in the CD23expression assay described in example 5).

The DNA sequence of the variable region of the light chain of MAb 6-2 ispresented in SEQ ID NO:5, and the encoded amino acid sequence ispresented in SEQ ID NO:6. The DNA sequence for the variable region ofthe heavy chain of MAb 6-2 is presented as SEQ ID NO:7, and the encodedamino acid sequence is presented in SEQ ID NO:8. Antibodies of thepresent invention include, but are not limited to, monoclonal antibodiesthat comprise, in their light chain, residues 1 to 107 of SEQ ID NO:6;and antibodies that additionally or alternatively comprise, in theirheavy chain, residues 1 to 118 of SEQ ID NO:8.

Complementarity determining regions (CDRs) of a given antibody may beidentified using the system described by Kabat et al. in Sequences ofProteins of Immunological Interest, 5^(th) Ed., US Dept. of Health andHuman Services, PHS, NIH, NIH Publication no. 91-3242, 1991). Particularembodiments of antibodies of the present invention comprise, within thevariable region of their light chain, at least one of thecomplementarity determining regions (CDRs), or hypervariable regions,found in the light chain of 6-2. CDRs of 6-2 are discussed in example 6.Thus, among the antibodies provided herein are those comprising from oneto all three of the following sequences in the light chain variableregion: amino acid residues 24-35 of SEQ ID NO:6; residues 51-57 of SEQID NO:6; and residues 90-97 of SEQ ID NO:6. Particular antibodiesprovided herein comprise, within the variable region of their heavychain, at least one of the CDRs found in the heavy chain of 6-2. Thus,among the antibodies provided herein are those comprising from one toall three of the following sequences in the heavy chain variable region:residues 31-35; residues 50-66; and residues 99-107 of SEQ ID NO:8.

Particular monoclonal antibodies of the invention are selected from thegroup consisting of MAb 12B5; a Mab that is cross-reactive with 12B5; aMAb that binds to the same epitope as 12B5; a MAb that competes with12B5 for binding to a cell that expresses human IL-4R; a MAb thatpossesses a biological activity of 12B5; and an antigen-binding fragmentof any of the foregoing antibodies. In one embodiment, the antibody hasa binding affinity for human IL-4R that is substantially equivalent tothe binding affinity of 12B5 for human IL-4R. MAb 12B5 is an IgG1antibody. MAbs of other isotypes, derived from 12B5, also areencompassed by the present invention. In particular embodiments, theisotype of the MAb is IgG1, IgG4, or IgM. Hybridoma cell lines thatproduce any such monoclonal antibodies also are provided by the presentinvention.

One example of a biological activity of 12B5 is the ability to functionas both an IL-4 antagonist and an IL-13 antagonist. In one embodiment, aMAb of the invention possesses IL-4-blocking activity substantiallyequivalent to that of 12B5, and possesses IL-13-blocking activitysubstantially equivalent to that of 12B5. Such activity may be measuredin any suitable conventional assay (e.g., as measured in the CD23expression assay described in example 5).

IgG4 monoclonal antibodies derived from 12B5 are provided herein.Another embodiment is directed to IgM monoclonal antibodies derived from12B5. Procedures for switching (altering) the subclass or isotype of anantibody are known in the pertinent field. Such procedures may involve,for example, recombinant DNA technology, whereby DNA encoding antibodypolypeptide chains that confer the desired subclass is substituted forDNA encoding the corresponding polypeptide chain of the parent antibody.

The DNA sequence of the variable region of the light chain of MAb 12B5is presented in SEQ ID NO:9, and the encoded amino acid sequence ispresented in SEQ ID NO:10. The DNA sequence for the variable region ofthe heavy chain of MAb 12B5 is presented as SEQ ID NO:11, and theencoded amino acid sequence is presented in SEQ ID NO:12. Antibodies ofthe present invention include, but are not limited to, monoclonalantibodies that comprise, in their light chain, residues 1 to 109 of SEQID NO:10; and antibodies that additionally or alternatively comprise, intheir heavy chain, residues 1 to 115 of SEQ ID NO:12.

Particular embodiments of antibodies of the present invention comprise,within the variable region of their light chain, at least one of thecomplementarity determining regions (CDRs), or hypervariable regions,found in the light chain of 12B5. CDRs of 12B5 are discussed in example8. Thus, among the antibodies provided herein are those comprising fromone to all three of the following sequences in the light chain variableregion: amino acid residues 24-35 of SEQ ID NO:10; residues 51-57 of SEQID NO:10; and residues 90-99 of SEQ ID NO:10. Particular antibodiesprovided herein comprise, within the variable region of their heavychain, at least one of the CDRs found in the heavy chain of 12B5. Thus,among the antibodies provided herein are those comprising from one toall three of the following sequences in the heavy chain variable region:residues 31-35; residues 50-65; and residues 98-104 of SEQ ID NO:12.

Particular monoclonal antibodies of the invention are selected from thegroup consisting of MAb 27A1; a Mab that is cross-reactive with 27A1; aMAb that binds to the same epitope as 27A1; a MAb that competes with27A1 for binding to a cell that expresses human IL-4R; a MAb thatpossesses a biological activity of 27A1; and an antigen-binding fragmentof any of the foregoing antibodies. In one embodiment, the antibody hasa binding affinity for human IL-4R that is substantially equivalent tothe binding affinity of 27A1 for human IL-4R. 27A1 is an IgG1 antibody.MAbs of other isotypes, derived from 27A1, also are encompassed by thepresent invention. Hybridoma cell lines that produce any such monoclonalantibodies also are provided by the present invention.

One example of a biological activity of 27A1 is the ability to functionas both an IL-4 antagonist and an IL-13 antagonist. In one embodiment, aMAb of the invention possesses IL-4-blocking activity substantiallyequivalent to that of 27A1; and possesses IL-13-blocking activitysubstantially equivalent to that of 27A1. Such activity may be measuredin any suitable conventional assay (e.g., as measured in the CD23expression assay described in example 5).

The DNA sequence of the variable region of the light chain of MAb 27A1is presented in SEQ ID NO:13, and the encoded amino acid sequence ispresented in SEQ ID NO:14. The DNA sequence for the variable region ofthe heavy chain of MAb 27A1 is presented as SEQ ID NO:15, and theencoded amino acid sequence is presented in SEQ ID NO:16. Antibodies ofthe present invention include, but are not limited to, monoclonalantibodies that comprise, in their light chain, residues 1 to 109 of SEQID NO:14; and antibodies that additionally or alternatively comprise, intheir heavy chain, residues 1 to 116 of SEQ ID NO:16.

Particular embodiments of antibodies of the present invention comprise,within the variable region of their light chain, at least one of thecomplementarity determining regions (CDRs), or hypervariable regions,found in the light chain of 27A1. CDRs of 27A1 are discussed in example9. Thus, among the antibodies provided herein are those comprising fromone to all three of the following sequences in the light chain variableregion: amino acid residues 24-35 of SEQ ID NO:14; residues 51-57 of SEQID NO:14; and residues 90-99 of SEQ ID NO:14. Particular antibodiesprovided herein comprise, within the variable region of their heavychain, at least one of the CDRs found in the heavy chain of 27A1. Thus,among the antibodies provided herein are those comprising from one toall three of the following sequences in the heavy chain variable region:residues 31-35; residues 50-66; and residues 99-105 of SEQ ID NO:16.

Particular monoclonal antibodies of the invention are selected from thegroup consisting of MAb 5A1; a Mab that is cross-reactive with 5A1; aMAb that binds to the same epitope as 5A1; a MAb that competes with 5A1for binding to a cell that expresses human IL-4R; a MAb that possesses abiological activity of 5A1; and an antigen-binding fragment of any ofthe foregoing antibodies. In one embodiment, the antibody has a bindingaffinity for human IL-4R that is substantially equivalent to the bindingaffinity of 5A1 for human IL-4R. 5A1 is an IgG1 antibody. MAbs of otherisotypes, derived from 5A1, also are encompassed by the presentinvention. Hybridoma cell lines that produce any such monoclonalantibodies also are provided by the present invention.

One example of a biological activity of 5A1 is the ability to functionas both an IL-4 antagonist and an IL-13 antagonist. In one embodiment, aMAb of the invention possesses IL-4-blocking activity substantiallyequivalent to that of 5A1; and possesses IL-13-blocking activitysubstantially equivalent to that of 5A1. Such activity may be measuredin any suitable conventional assay (e.g., as measured in the CD23expression assay described in example 5).

The DNA sequence of the variable region of the light chain of MAb 5A1 ispresented in SEQ ID NO:17, and the encoded amino acid sequence ispresented in SEQ ID NO:18. The DNA sequence for the variable region ofthe heavy chain of MAb 5A1 is presented as SEQ ID NO:19, and the encodedamino acid sequence is presented in SEQ ID NO:20. Antibodies of thepresent invention include, but are not limited to, monoclonal antibodiesthat comprise, in their light chain, residues 1 to 107 of SEQ ID NO:18;and antibodies that additionally or alternatively comprise, in theirheavy chain, residues 1 to 123 of SEQ ID NO:20.

Particular embodiments of antibodies of the present invention comprise,within the variable region of their light chain, at least one of thecomplementarity determining regions (CDRs), or hypervariable regions,found in the light chain of 5A1. CDRs of 5A1 are discussed in example 9.Thus, among the antibodies provided herein are those comprising from oneto all three of the following sequences in the light chain variableregion: amino acid residues 24-34 of SEQ ID NO:18; residues 50-56 of SEQID NO:18; and residues 89-97 of SEQ ID NO:18. Particular antibodiesprovided herein comprise, within the variable region of their heavychain, at least one of the CDRs found in the heavy chain of 5A1. Thus,among the antibodies provided herein are those comprising from one toall three of the following sequences in the heavy chain variable region:residues 31-35; residues 50-65; and residues 98-112 of SEQ ID NO:20.

Particular monoclonal antibodies of the invention are selected from thegroup consisting of MAb 63; a Mab that is cross-reactive with MAb 63; aMAb that binds to the same epitope as 63; a MAb that competes with 63for binding to a cell that expresses human IL-4R; a MAb that possesses abiological activity of 63; and an antigen-binding fragment of any of theforegoing antibodies. In one embodiment, the antibody has a bindingaffinity for human IL-4R that is substantially equivalent to the bindingaffinity of 63 for human IL-4R. MAb 63 is an IgM antibody. MAbs of otherisotypes, derived from 63, also are encompassed by the presentinvention. Hybridoma cell lines that produce any such monoclonalantibodies also are provided by the present invention.

One example of a biological activity of 63 is the ability to function asboth an IL-4 antagonist and an IL-13 antagonist. In one embodiment, aMAb of the invention possesses IL-4-blocking activity substantiallyequivalent to that of 63; and possesses IL-13-blocking activitysubstantially equivalent to that of 63. Such activity may be measured inany suitable conventional assay (e.g., as measured in the CD23expression assay described in example 5).

The DNA sequence of the variable region of the light chain of MAb 63 ispresented in SEQ ID NO:21, and the encoded amino acid sequence ispresented in SEQ ID NO:22. The DNA sequence for the variable region ofthe heavy chain of MAb 63 is presented as SEQ ID NO:23, and the encodedamino acid sequence is presented in SEQ ID NO:24. Antibodies of thepresent invention include, but are not limited to, monoclonal antibodiesthat comprise, in their light chain, residues 1 to 107 of SEQ ID NO:22;and antibodies that additionally or alternatively comprise, in theirheavy chain, residues 1 to 117 of SEQ ID NO:24.

Particular embodiments of antibodies of the present invention comprise,within the variable region of their light chain, at least one of thecomplementarity determining regions (CDRs), or hypervariable regions,found in the light chain of 63. CDRs of 63 are discussed in example 9.Thus, among the antibodies provided herein are those comprising from oneto all three of the following sequences in the light chain variableregion: amino acid residues 24-34 of SEQ ID NO:22; residues 50-56 of SEQID NO:22; and residues 89-97 of SEQ ID NO:22. Particular antibodiesprovided herein comprise, within the variable region of their heavychain, at least one of the CDRs found in the heavy chain of 63. Thus,among the antibodies provided herein are those comprising from one toall three of the following sequences in the heavy chain variable region:residues 31-35; residues 50-66; and residues 99-106 of SEQ ID NO:24.

Particular monoclonal antibodies of the invention are selected from thegroup consisting of MAb 1B7; a Mab that is cross-reactive with 1B7; aMAb that binds to the same epitope as 1B7; a MAb that competes with 1B7for binding to a cell that expresses human IL-4R; a MAb that possesses abiological activity of 1B7; and an antigen-binding fragment of any ofthe foregoing antibodies. In one embodiment, the antibody has a bindingaffinity for human IL-4R that is substantially equivalent to the bindingaffinity of 1B7 for human IL-4R. MAbs of other isotypes, derived from1B7, also are encompassed by the present invention. Hybridoma cell linesthat produce any such monoclonal antibodies also are provided by thepresent invention.

One example of a biological activity of 1B7 is the ability to functionas both an IL-4 antagonist and an IL-13 antagonist. In one embodiment, aMAb of the invention possesses IL-4-blocking activity substantiallyequivalent to that of 1B7; and possesses IL-13-blocking activitysubstantially equivalent to that of 1B7. Such activity may be measuredin any suitable conventional assay (e.g., as measured in the CD23expression assay described in example 5).

MAb 1B7 was derived from MAb 63. The amino acid sequence of the heavychain of MAb 1B7 is identical to that of MAb 63. The only differencesbetween the two antibodies are in the light chain. The DNA sequence ofthe variable region of the light chain of MAb 1B7 is presented in SEQ IDNO:25, and the encoded amino acid sequence is presented in SEQ ID NO:26.The DNA sequence for the variable region of the heavy chain of MAb 1B7is presented as SEQ ID NO:23, and the encoded amino acid sequence ispresented in SEQ ID NO:24 (same as for MAb 63). Antibodies of thepresent invention include, but are not limited to, monoclonal antibodiesthat comprise, in their light chain, residues 1 to 107 of SEQ ID NO:26;and antibodies that additionally or alternatively comprise, in theirheavy chain, residues 1 to 117 of SEQ ID NO:24.

Particular embodiments of antibodies of the present invention comprise,within the variable region of their light chain, at least one of thecomplementarity determining regions (CDRs), or hypervariable regions,found in the light chain of 1B7. CDRs of 1B7 are discussed in example 9.Thus, among the antibodies provided herein are those comprising from oneto all three of the following sequences in the light chain variableregion: amino acid residues 24-34 of SEQ ID NO:26; residues 50-56 of SEQID NO:26; and residues 89-97 of SEQ ID NO:26. Particular antibodiesprovided herein comprise, within the variable region of their heavychain, at least one of the CDRs found in the heavy chain of 1B7. Thus,among the antibodies provided herein are those comprising from one toall three of the following sequences in the heavy chain variable region:residues 31-35; residues 50-66; and residues 99-106 of SEQ ID NO:24.

Derivatives of monoclonal antibodies directed against IL-4R may beprepared, and screened for desired properties, by any of a number ofknown techniques. Certain of the techniques involve isolating DNAencoding a polypeptide chain (or portion thereof) of a MAb of interest,and manipulating the DNA through recombinant DNA technology. The DNA maybe fused to another DNA of interest, or altered (e.g., by mutagenesis orother conventional techniques) to add, delete, or substitute one or moreamino acid residues, for example.

DNA encoding antibody polypeptides (e.g., heavy or light chain, variableregion only or full length) may be isolated from B-cells of mice thathave been immunized with IL-4R. The DNA may be isolated by conventionalprocedures such as polymerase chain reaction (PCR). Phage display isanother example of a known technique whereby derivatives of antibodiesmay be prepared. In one approach, polypeptides that are components of anantibody of interest are expressed in any suitable recombinantexpression system, and the expressed polypeptides are allowed toassemble to form antibody molecules.

Single chain antibodies may be formed by linking heavy and light chainvariable region (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable regionpolypeptides (V_(L) and V_(H)). The resulting antibody fragments canform dimers or trimers, depending on the length of a flexible linkerbetween the two variable domains (Kortt et al., Protein Engineering10:423, 1997). Techniques developed for the production of single chainantibodies include those described in U.S. Pat. No. 4,946,778; Bird(Science 242:423, 1988); Huston et al. (Proc. Natl. Acad. Sci. USA85:5879, 1988); and Ward et al. (Nature 334:544, 1989). Single chainantibodies derived from antibodies provided herein (including but notlimited to scFvs derived from MAbs 6-2, 12B5, 63, 1B7, 5A1, and 27A1)are encompassed by the present invention.

Techniques are known for deriving an antibody of a different subclass orisotype from an antibody of interest, i.e., subclass switching. Thus,IgG1 or IgG4 monoclonal antibodies may be derived from an IgM monoclonalantibody, for example, and vice versa. Such techniques allow thepreparation of new antibodies that possess the antigen-bindingproperties of a given antibody (the parent antibody), but also exhibitbiological properties associated with an antibody isotype or subclassdifferent from that of the parent antibody. Recombinant DNA techniquesmay be employed. Cloned DNA encoding particular antibody polypeptidesmay be employed in such procedures, e.g., DNA encoding the constantregion of an antibody of the desired isotype.

In particular embodiments, antibodies raised against IL-4R have abinding affinity (Ka) for IL-4R of at least 1×10⁸. In other embodiments,the antibodies exhibit a Ka of at least 1×10⁹ or at least 1×10¹⁰.

PEGylated derivatives of antibodies (modified with polyethylene glycol)also are contemplated, and may be prepared by conventional techniques.Also provided herein are conjugates comprising a detectable (e.g.,diagnostic) or therapeutic agent, attached to an antibody directedagainst IL-4R. Examples of such agents are well known, and include butare not limited to diagnostic radionuclides, therapeutic radionuclides,and cytotoxic drugs. The conjugates find use in in vitro or in vivoprocedures.

Particular embodiments of the invention are directed to novel nucleicacid molecules and polypeptides. DNA and amino acid sequence informationhas been determined for polypeptides that are components of certainantibodies of the present invention, as discussed in examples 6, 8, and9 below. Among the nucleic acids of the present invention is isolatedDNA comprising a nucleotide sequence selected from the group consistingof the nucleotide sequence presented in SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, and SEQ ID NO:25. Among thepolypeptides of the present invention is a purified polypeptidecomprising an amino acid sequence selected from the group consisting ofthe amino acid sequence presented in SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26. For in vivo use,the polypeptides advantageously are purified. A polypeptide may bepurified individually, or in the form of a purified antibody of whichthe polypeptide is a component.

Further examples of IL-4 antagonists are antibodies that bind IL-4 andinhibit the binding of IL-4 to cell surface receptors. Such antibodiesmay be prepared, and screened to identify those that are blockingantibodies, by conventional procedures. Antigen-binding fragments ofsuch antibodies find use as antagonists, as do humanized or geneticallyengineered derivatives thereof.

Examples of procedures for preparing antibodies directed against humanIL-4 (including monoclonal antibodies), assays by which blockingantibodies are identified, and techniques for generating humanized orgenetically engineered derivatives of anti-IL-4 antibodies, aredescribed in U.S. Pat. Nos. 5,041,381, 5,863,537, 5,928,904, and5,676,940, which are hereby incorporated by reference. Further examplesof antibodies that may be employed as IL-4 antagonists are described inWO 91/09059, also incorporated by reference herein.

Other Antagonists

Any compound that functions as an IL-4 antagonist and is suitable foradministration in accordance with the methods of the present inventionmay be employed. Antagonists need not completely abolish IL-4-inducedbiological activity to be useful. Rather, a given antagonist may reducea biological activity of IL-4.

Derivatives, mutants/muteins, and other variants of IL-4 that functionas IL-4 antagonists may be employed. Peptides (which may or may not bemuteins) derived from IL-4 that bind to an IL-4R without inducingtransduction of a biological signal find use herein. Such peptidesfunction as inert blockers, interfering with the binding of biologicallyactive endogenous IL-4 to cell surface receptors. IL-4-induced signaltransduction thereby is inhibited. Muteins or other antagonists thatinduce a biological response at a reduced level or to a lesser degree,compared to the response induced by native IL-4, also find use as IL-4antagonists.

Further examples of IL-4 antagonists, including IL-4 muteins, andprocedures for preparation thereof are described in Muller et al., J.Mol. Biol., 237:423-436, 1994; U.S. Pat. No. 6,028,176, and U.S. Pat.No. 5,723,118, which are each incorporated by reference herein.

Other options are antisense molecules (oligonucleotides) that inhibitexpression of IL-4. Alternatively, the antisense molecule may suppressexpression of other molecules involved in IL-4-induced signaltransduction.

Any suitable assay, including in vitro assays, can be utilized todetermine whether a given compound inhibits an IL-4-induced biologicalactivity. An antagonist may be assayed for the ability to inhibit³H-thymidine incorporation in cells that normally proliferate inresponse to IL-4.

An alternative involves use of conventional binding assay techniques totest an antagonist for the ability to inhibit binding of IL-4 to cellsexpressing native or recombinant IL-4 receptors. For use in such assays,recombinant human IL-4 can be expressed and purified as described inU.S. Pat. No. 5,017,691, hereby incorporated by reference herein, or inPark et al., J. Exp. Med. 166:476 (1987). The purified protein may belabeled with a detectable agent (e.g., radiolabeled) by any of a numberof conventional techniques. A commercially available enzymobeadradioiodination reagent (BioRad) may be employed in radiolabeling IL-4with ¹²⁵I for example.

The ability of an IL-4 antagonist to inhibit IL-4-induced damage toepithelium, such as lung epithelium or intestinal epithelium (which mayresult in loss of barrier function), may be confirmed in any of a numberof suitable assays. Among the suitable assay techniques are thosedescribed in example 7 below.

Therapeutic Methods and Administration of Antagonists

Methods provided herein comprise administering an IL-4 antagonist to apatient, thereby reducing an IL-4-induced biological response that playsa role in a particular condition. In particular embodiments, methods ofthe invention involve contacting endogenous IL-4 with an IL-4antagonist, e.g., in an ex vivo procedure.

Treatment encompasses alleviation of at least one symptom of a disorder,or reduction of disease severity, and the like. An antagonist need noteffect a complete “cure”, or eradicate every symptom or manifestation ofa disease, to constitute a viable therapeutic agent. As is recognized inthe pertinent field, drugs employed as therapeutic agents may reduce theseverity of a given disease state, but need not abolish everymanifestation of the disease to be regarded as useful therapeuticagents. One embodiment of the invention is directed to a methodcomprising administering to a patient an IL-4 antagonist in an amountand for a time sufficient to induce a sustained improvement overbaseline of an indicator that reflects the severity of the particulardisorder.

Antibodies that inhibit the binding of both IL-4 and IL-13 to cells arediscussed herein. A method for suppressing IL-4-induced andIL-13-induced activities in humans comprises administering an effectiveamount of such an antibody. Conditions induced by IL-4 or by IL-13, orby both cytokines, thus may be treated.

As is understood in the pertinent field, antagonists are administered toa patient in a manner appropriate to the indication. Antagonists may beadministered by any suitable technique, including but not limited toparenterally, topically, or by inhalation. If injected, the antagonistcan be administered, for example, via intra-articular, intravenous,intramuscular, intralesional, intraperitoneal or subcutaneous routes, bybolus injection, or continuous infusion. Localized administration, e.g.at a site of disease or injury is contemplated, as are transdermaldelivery and sustained release from implants. Delivery by inhalationincludes, for example, nasal or oral inhalation, use of a nebulizer,inhalation of the antagonist in aerosol form, and the like. Otheralternatives include eyedrops; oral preparations including pills,syrups, lozenges or chewing gum; and topical preparations such aslotions, gels, sprays, and ointments.

Use of IL-4 antagonists in ex vivo procedures is contemplated. Forexample, a patient's blood (bodily fluid containing IL-4) may becontacted with an antagonist that binds IL-4 ex vivo, thereby reducingthe amount of IL-4 in the fluid when returned to the patient. Theantagonist may be bound to a suitable insoluble matrix or solid supportmaterial.

Advantageously, antagonists are administered in the form of acomposition comprising at least one IL-4 antagonist and one or moreadditional components such as a physiologically acceptable carrier,excipient or diluent. The present invention provides such compositionscomprising an effective amount of an IL-4 antagonist, for use in themethods provided herein.

The compositions contain antagonist(s) in any of the forms describedherein. The antagonist may be a whole antibody or an antigen-bindingfragment or engineered derivative thereof, for example. For compositionscontaining an IL-4 receptor, the receptor may be any of the fragments,variants, or oligomers of the protein of SEQ ID NO:2 described herein,for example.

Compositions may, for example, comprise an antagonist together with abuffer, antioxidant such as ascorbic acid, low molecular weightpolypeptide (such as those having fewer than 10 amino acids), protein,amino acid, carbohydrate such as glucose, sucrose or dextrins, chelatingagents such as EDTA, glutathione, and other stabilizers and excipients.Neutral buffered saline or saline mixed with conspecific serum albuminare examples of appropriate diluents. In accordance with appropriateindustry standards, preservatives such as benzyl alcohol may also beadded. The composition may be formulated as a lyophilizate usingappropriate excipient solutions (e.g., sucrose) as diluents. Suitablecomponents are nontoxic to recipients at the dosages and concentrationsemployed. Further examples of components that may be employed inpharmaceutical formulations are presented in Remington's PharmaceuticalSciences, 16^(th) Ed., Mack Publishing Company, Easton, Pa., 1980.

Kits for use by medical practitioners include an IL-4 antagonist and alabel or other instructions for use in treating any of the conditionsdiscussed herein. The kit preferably includes a sterile preparation ofone or more IL-4 antagonists, which may be in the form of a compositionas disclosed above, and may be in one or more vials.

Dosages and the frequency of administration may vary according to suchfactors as the route of administration, the particular antagonistemployed, the nature and severity of the disease to be treated, whetherthe condition is acute or chronic, and the size and general condition ofthe patient. Appropriate dosages can be determined by procedures knownin the pertinent art, e.g. in clinical trials that may involve doseescalation studies.

An antagonist may be administered once, or repeatedly. In particularembodiments, the antagonist is administered over a period of at least amonth or more, e.g., for one, two, or three months or even indefinitely.For treating chronic conditions, long-term treatment is generally mosteffective. However, for treating acute conditions, administration forshorter periods, e.g. from one to six weeks, may be sufficient. Ingeneral, the antagonist is administered until the patient manifests amedically relevant degree of improvement over baseline for the chosenindicator or indicators.

Particular embodiments of the present invention involve administering anantagonist at a dosage of from about 1 ng/kg/day to about 10 mg/kg/day,more preferably from about 500 ng/kg/day to about 5 mg/kg/day, and mostpreferably from about 5 ug/kg/day to about 2 mg/kg/day, to a patient. Inadditional embodiments, an antagonist such as a soluble human IL-4Rpolypeptide is administered to adults one time per week, two times perweek, or three or more times per week, to treat the medical disordersdisclosed herein. If injected, the effective amount of antagonist peradult dose may range from 1-20 mg/m², and preferably is about 5-12mg/m². Alternatively, a flat dose may be administered; the amount mayrange from 5-100 mg/dose. One range for a flat dose is about 20-30 mgper dose. In one embodiment of the invention, a flat dose of 25 mg/doseis repeatedly administered by injection. If a route of administrationother than injection is used, the dose is appropriately adjusted inaccordance with standard medical practices. One example of a therapeuticregimen involves injecting a dose of about 20-30 mg of IL-4R or otherantagonist one to three times per week over a period of at least threeweeks, though treatment for longer periods may be necessary to inducethe desired degree of improvement. For pediatric patients (age 4-17),one suitable regimen involves the subcutaneous injection of 0.4 mg/kg,up to a maximum dose of 25 mg of IL-4R, administered two or three timesper week.

Particular embodiments of the methods provided herein involvesubcutaneous injection of from 0.5 mg to 10 mg, preferably from 3 to 5mg, of a soluble IL-4R, once or twice per week. Another embodiment isdirected to pulmonary administration (e.g., by nebulizer) of 3 or moremg of a soluble IL-4R once a week.

Examples of therapeutic regimens provided herein comprise subcutaneousinjection of a soluble human IL-4R once a week, at a dose of 1.5 to 3mg, to treat pulmonary sarcoidosis, minimal change nephrosis, autoimmuneuveitis, sickle cell crisis, Churg-Strauss syndrome, Sjogren's syndrome,autoimmune lymphoproliferative syndrome, pre-eclampsia, autoimmunehemolytic anemia, Barrett's esophagus, Grave's Disease, KawasakiDisease, and cavitary buberculosis. Weekly administration of IL-4R iscontinued until symptoms subside. Treatment may resume as needed, or,alternatively, maintenance doses may be administered.

An antagonist is administered to the patient in an amount and for a timesufficient to induce an improvement, preferably a sustained improvement,in at least one indicator that reflects the severity of the disorderthat is being treated. Various indicators that reflect the extent of thepatient's illness may be assessed for determining whether the amount andtime of the treatment is sufficient. Such indicators include, forexample, clinically recognized indicators of disease severity, symptoms,or manifestations of the disorder in question. In most instances, animprovement is considered to be sustained if the patient exhibits theimprovement on at least two occasions separated by two to four weeks.The degree of improvement generally is determined by the patient'sphysician, who may make this determination based on signs or symptoms,and who may also employ questionnaires that are administered to thepatient, such as quality-of-life questionnaires developed for a givendisease.

As one example, in treating benign prostate hyperplasia, an IL-4inhibitor is administered to the patient in an amount and for a timeeffective in scar regression or complete healing. Maintenance doses maybe given or treatment resumed as needed.

Elevated levels of IL-4 are associated with a number of disorders, asdiscussed above. Patients with a given disorder may be screened, toidentify those individuals who have elevated IL-4 levels, or to identifythose with an elevated TH2-type immune response, thereby identifying thepatients who may benefit most from treatment with an IL-4 antagonist.Thus, treatment methods provided herein optionally comprise a first stepof measuring a patient's IL-4 level. An IL-4 antagonist may beadministered to a patient in whom IL-4 levels are elevated above normal.Alternatively or additionally, a patient may be pre-screened todetermine whether the patient has an elevated TH2-type immune response,prior to administration of antagonist(s) against one or more TH2-typecytokines.

A patient's levels of IL-4 (and, optionally, of other TH2-typecytokines) may be monitored during and/or after treatment with an IL-4antagonist, to detect reduction in the levels of the cytokines. For somedisorders, the incidence of elevated IL-4 levels, and the balancebetween TH1-type and TH2-type immune responses, may vary according tosuch factors as the stage of the disease or the particular form of thedisease. Known techniques may be employed for measuring IL-4 levels,e.g., in a patient's serum, and for assessing TH2-type immune responses.Cytokine levels in blood samples may be measured by ELISA, for example.

Particular embodiments of methods and compositions of the inventioninvolve the use of two or more different IL-4 antagonists. In furtherembodiments, IL-4 antagonist(s) are administered alone or in combinationwith other agents useful for treating the condition with which thepatient is afflicted. Examples of such agents include both proteinaceousand non-proteinaceous drugs. When multiple therapeutics areco-administered, dosages may be adjusted accordingly, as is recognizedin the pertinent art. “Co-administration” and combination therapy arenot limited to simultaneous administration, but include treatmentregimens in which an IL-4 antagonist is administered at least onceduring a course of treatment that involves administering at least oneother therapeutic agent to the patient.

Examples of other agents that may be co-administered with IL-4antagonists are other antibodies, cytokines, or cytokine receptors,which are chosen according to the particular condition to be treated.Alternatively, non-proteinaceous drugs that are useful in treating oneof the particular conditions discussed above may be co-administered withan IL-4 antagonist.

For treating IgE-mediated conditions, an IL-4 antagonist may beco-administered with an IgE antagonist. One example is an anti-IgEantibody. Humanized anti-IgE monoclonal antibodies are described inPresta et al. (J. Immunol. 151(5):2623-2632, 1993) and Adelroth et al.(J. Allergy Clin. Immunol. 106(2):253-259, 2000), for example.

IL-4 antagonists may be co-administered with an IL-5 antagonist, whichmay be a molecule that interferes with the binding of IL-5 to an IL-5receptor, such as an anti-IL-5 antibody (e.g., a human or humanizedanti-IL-5 monoclonal antibody), or a receptor such as a soluble humanIL-5 receptor polypeptide. IL-5 has been implicated as playing a role inmediating allergic responses. Thus, administration of antagonist(s) ofIL-4 and IL-5 is contemplated for treatment of allergic reactions,including but not limited to allergic asthma.

IL-4 antagonists may be employed in conjunction with other agent(s) intreating the particular IL-4-induced conditions discussed above. Forexample, drugs currently employed in treating the conditions may beco-administered with one or more IL-4 antagonists.

For treating asthma, an IL-4 antagonist may be co-administered withother anti-asthma medications, such as inhaled corticosteroids, betaagonists, leukotriene antagonists, xanthines, fluticasone, salmeterol,albuterol, non-steroidal agents such as cromolyn, and the like. IL-4antagonists may be co-administered with other anti-allergy medicationsto treat allergic reactions.

One embodiment of the present invention is directed to co-administrationof an IL-4 antagonist (such as a soluble human IL-4R) and fluticasoneand salmeterol to treat a disorder such as asthma. Compositionscomprising an IL-4 inhibitor (e.g., soluble human IL-4R), fluticasone,and salmeterol are provided herein. Advair Diskus (Glaxo Wellcome)comprises fluticasone propionate and salmeterol xinafoate. For treatingasthma, Advair Diskus and the IL-4 antagonist preferably are deliveredby inhalation.

Another example of combination therapy comprises co-administration of anIL-4 antagonist and an IL-9 antagonist to a patient who has asthma. Anysuitable IL-9 antagonist may be employed, such as an IL-9 receptor(preferably a soluble form thereof), an antibody that interferes withbinding of IL-9 to a cell surface receptor (wherein the antibody may beraised against IL-9 or an IL-9 receptor polypeptide), or anothercompound that inhibits IL-9-induced biological activity. IL-9 receptorsinclude those described in WO 93/18047 and U.S. Pat. Nos. 5,789,237 and5,962,269, which are hereby incorporated by reference herein.

In an additional embodiment of combination therapy, a method fortreating ulcerative colitis comprises co-administration of at least oneIL-4 antagonist and at least one IL-1 antagonist. Examples of IL-1antagonists include type I IL-1 receptor, type II IL-1 receptor, IL-1receptor antagonist (IL-1Ra), antagonistic (blocking) antibodiesdirected against IL-1, and antagonistic antibodies directed against anIL-1 receptor. Various forms of the receptors may be employed, such asfragments, variants and fusions analogous to those described above forIL-4 receptor. A preferred IL-1 antagonist is a soluble form of type IIIL-1 receptor, which is described in U.S. Pat. No. 5,350,683, herebyincorporated by reference herein.

One method of the present invention comprises co-administering IL-4antagonist(s) and IL-13 antagonist(s) to a patient who has minimalchange nephrosis. Alternative embodiments involve administering IL-4antagonist(s) alone, or IL-13 antagonist(s) alone, to a minimal changenephrosis patient. The IL-4 antagonists(s) and/or IL-13 antagonist(s)may be administered to reduce severity of the disease.

Another method provided herein is a method for treating various allergicinflammatory conditions, comprising co-administering IL-4 antagonist(s)and IL-13 antagonist(s). Conditions such as asthma, allergies, andchronic lung diseases such as cystic fibrosis and chronic obstructivepulmonary disease are treated by such a method.

Any suitable IL-13 antagonist may be employed, including but not limitedto IL-13 receptors (preferably soluble forms thereof), IL-13 receptorantagonists, antibodies directed against IL-13 or an IL-13 receptor,other proteins that interfere with the binding of IL-13 to an IL-13receptor, and compounds that inhibit IL-13-mediated signal transduction.IL-13 receptors and heterodimers comprising IL-13R polypeptides ascomponents thereof are described above. Antibodies that are raisedagainst IL-4R may be screened for the ability to also function as IL-13antagonists, as discussed above.

A method for treating or preventing a condition characterized by reducedepithelial barrier function comprises co-administering IL-4antagonist(s) and one or more IL-13 antagonists. Such conditions arediscussed above. In one embodiment, the condition is asthma. Particularembodiments are directed to co-administering one or more IL-4antagonists and one or more IL-13 antagonists to a patient having acondition involving reduction of lung epithelial barrier function orintestinal epithelial barrier function, wherein both IL-4 and IL-13 playa role in the reduced barrier function. The method thus inhibits bothIL-4-induced reduction of barrier function and IL-13-induced reductionof barrier function. The adverse effect of IL-13 on lung and intestinalepithelial barrier function can be confirmed using assay techniques suchas those described in example 7 below. (See also Zund et al., J. Biol.Chem. 271(13):7460-7464, 1996.)

Another method provided herein comprises co-administering IL-4antagonist(s) and interferon-γ (IFN-γ) to a patient having a conditioninvolving reduction of lung epithelial barrier function. Optionally,such a method further comprises co-administering one or more IL-13antagonists to the patient (i.e., co-administering an IL-4 antagonist,IFN-γ, and an IL-13 antagonist). Other methods comprise administeringIFN-γ as a single agent, or co-administering IFN-γ and an IL-13antagonist, to a patient having a condition involving reduction of lungepithelial barrier function. In one embodiment, the patient has asthma.For treating asthma, the IL-4 antagonist, IFN-γ, and/or IL-13 antagonistpreferably are administered by inhalation.

One method provided herein for treating asthma comprises administeringan IL-4 antagonist and interferon-γ to a human who has asthma. Anothermethod for treating asthma comprises co-administering an IL-4antagonist, IFN-γ, and an IL-13 antagonist to a human who has asthma. Inone embodiment, IFN-γ is co-administered to an asthmatic, together withan antibody that functions as an antagonist of both IL-4 and IL-13. Suchantibodies are described elsewhere herein.

A single agent may function as an IL-4 antagonist and an IL-13antagonist, as discussed above. As an example of such an agent, someantibodies raised against IL-4Rα may interfere with the binding of bothIL-4 and IL-13 receptor complexes, due to the shared IL-4Rα component insuch multi-subunit receptor complexes (discussed above). Thus, a singleagent may be employed in a method for inhibiting reduction of barrierfunction.

Antagonists may be co-administered with one or more leukotriene receptorantagonists to treat disorders such as allergic inflammatory diseases,e.g., asthma and allergies. Examples of leukotriene receptor antagonistsinclude but are not limited to montelukast, pranlukast, and zafirlukast.Drugs that function as 5-lipoxygenase inhibitors may be co-administeredwith an IL-4 antagonist to treat asthma.

Methods provided herein comprise administering one or more of thefollowing to Churg-Strauss Syndrome patients: IL-4 antagonist(s), IL-5antagonist(s), IL-13 antagonist(s) or IgE antagonist(s). One example ofsuch a method involves co-administering IL-4 antagonist(s) and IL-5antagonist(s) to a Churg-Strauss Syndrome patient. In anotherembodiment, IL-4 antagonist(s) and IgE antagonist(s) are co-administeredto the patient. In yet another embodiment, IL-4 antagonist(s) and IL-13antagonist(s) are co-administered to the patient.

The hormone relaxin may be co-administered with an IL-4 antagonist totreat scleroderma (systemic sclerosis), idiopathic pulmonary fibrosis,or any other disorder characterized by pulmonary fibrosis, such as theconditions involving fibrosis of the lung that are discussed above.Recombinant human relaxin is preferred for use in treating humans.

A method for treating benign prostate hyperplasia comprisesco-administering IL-4 antagonist(s) and one or more additionalanti-inflammatory agents. Examples of agents that inhibit inflammationinclude tumor necrosis factor (TNF) antagonists and IL-17 antagonists.

Any suitable IL-17 antagonist may be employed, including but not limitedto an IL-17 receptor (preferably soluble forms thereof), IL-17 receptorantagonists, antibodies directed against IL-17 or an IL-17 receptor,other proteins that interfere with the binding of IL-17 to an IL-17receptor, and compounds that inhibit IL-17-mediated signal transduction.An IL-17 receptor, including soluble forms thereof and oligomersthereof, is described in WO 96/29408, hereby incorporated by reference.An alternative method provided herein comprises administering an IL-17antagonist to treat a patient with benign prostate hyperplasia.

Likewise, any suitable TNF antagonist may be employed, including but notlimited to a TNF receptor (preferably soluble forms thereof), fusionproteins comprising a TNF receptor (or comprising the TNF-bindingportion of a TNF receptor), TNF receptor antagonists, antibodiesdirected against TNF or a TNF receptor, other proteins that interferewith the binding of TNF to a TNF receptor, and compounds that inhibitTNF-mediated signal transduction. Further examples of TNF inhibitors arethe drugs thalidomide and pentoxyfylline. The TNF receptor protein knownas p75 or p80 TNF-R preferably is employed. A preferred TNF antagonistis a soluble human TNF receptor (sTNF-R) in dimeric form, such as dimersof sTNF-R/Fc fusion proteins. One such dimer is etanercept (Enbrel®,Immunex Corporation, Seattle, Wash.). p75/p80 TNF-R, including solublefragments and other forms thereof, is described in WO 91/03553, herebyincorporated by reference herein.

In accordance with the present invention, an IL-4 antagonist isco-administered with a TNF antagonist to treat any condition in whichundesirable IL-4-induced and TNF-induced immune responses play a role,such as inflammation. One method provided herein comprisesco-administering an IL-4 antagonist and a TNF antagonist to a patientwith inflammatory bowel disease, Crohn's disease, or ulcerative colitis.Other embodiments are directed to a method comprising co-administeringan IL-4 antagonist and a TNF antagonist to a patient who has KawasakiDisease, autoimmune hemolytic anemia, autoimmune uveoretinitis,autoimmune lymphoproliferative syndrome, Sjogren's syndrome, chronicfatigue syndrome, or hepatotoxicity induced by a drug such asdiclofenac.

Another method provided herein comprises co-administering an IL-4antagonist and a TNF antagonist to a pregnant woman who has developedpre-eclampsia. Administration of the IL-4 antagonist and TNF-antagonistpreferably continues for the duration of the pregnancy.

Suitable dosages of etanercept (Enbrel®, Immunex Corporation, Seattle,Wash.) will vary according to the nature of the disease to be treated,disease severity, the size of the patient (e.g., adult or child), andother factors, as is recognized in the pertinent field. In oneembodiment of the methods provided herein, Enbrel® is administered twicea week by subcutaneous injection at a dose of from 1 to 25 mg. Oneembodiment of a pediatric dosage is 0.4 mg/kg. Particular methodsprovided herein comprise co-administration of an IL-4 antagonist andEnbrel® to a patient has autoimmune lymphoproliferative syndrome orSjogren's syndrome, wherein Enbrel® is given by subcutaneous injectionat a dose of from 1 to 25 mg.

For treating graft versus host disease, an IL-4 antagonist isco-administered with at least one of the following agents: a TNFantagonist, an IL-1 antagonist, steroids, or corticosteroids. The TNFinhibitor preferably is Enbrel®. A preferred IL-1 antagonist is asoluble form of type II IL-1 receptor, which is described in U.S. Pat.No. 5,350,683. In one embodiment, the GVHD is associated with (e.g.,develops subsequent to) bone marrow transplantation. An IL-4 antagonistmay be employed in combination with at least one of the above-listedagents, in methods for suppressing an immune response directed againsttransplanted cells, tissue, and/or alloantigen.

A number of cytokine antagonists and other agents/drugs are disclosedherein as being useful for combination therapy (e.g., co-administrationwith an IL-4 antagonist) in treating particular diseases. It is to beunderstood that such antagonists, agents, or drugs also find use assingle agents in treating those diseases. It also is to be understoodthat disclosure of methods involving administration of an antagonist toa particular cytokine, to treat a disease, encompasses administration ofone type of antagonist, and also encompasses administration of two ormore different antagonists for that cytokine, unless specifiedotherwise.

The following examples are offered by way of illustration, and not byway of limitation.

Example 1: Preparation of Monoclonal Antibodies

IL-4 receptor polypeptides may be employed as immunogens in generatingmonoclonal antibodies by conventional techniques, e.g., techniquesdescribed in U.S. Pat. No. 5,599,905, hereby incorporated by reference.It is recognized that polypeptides in various forms may be employed asimmunogens, e.g., full length proteins, fragments thereof, fusionproteins thereof such as Fc fusions, cells expressing the recombinantprotein on the cell surface, etc.

To summarize an example of such a procedure, an IL-4R immunogenemulsified in complete Freund's adjuvant is injected subcutaneously intoLewis rats, in amounts ranging from 10-100 μl. Three weeks later, theimmunized animals are boosted with additional immunogen emulsified inincomplete Freund's adjuvant and boosted every three weeks thereafter.Serum samples are periodically taken by retro-orbital bleeding ortail-tip excision for testing by dot-blot assay, ELISA (enzyme-linkedimmunosorbent assay), or inhibition of binding of ¹²⁵I-IL-4 to extractsof IL-4R-expressing cells. Following detection of an appropriateantibody titer, positive animals were given a final intravenousinjection of antigen in saline. Three to four days later, the animalsare sacrificed, splenocytes harvested, and fused to the murine myelomacell line AG8653. The resulting hybridoma cell lines are plated inmultiple microtiter plates in a HAT selective medium (hypoxanthine,aminopterin, and thymidine) to inhibit proliferation of non-fused cells,myeloma hybrids, and spleen cell hybrids.

Hybridoma clones thus generated are screened for reactivity with IL-4R.Initial screening of hybridoma supernatants utilizes an antibody captureand binding of partially purified ¹²⁵I-IL-4 receptor. Hybridomas thatare positive in this screening method are tested by a modified antibodycapture to detect hybridoma cells lines that are producing blockingantibody. Hybridomas that secrete a monoclonal antibody capable ofinhibiting ¹²⁵I-IL-4 binding to cells expressing IL-4R are thusdetected. Such hydridomas then are injected into the peritoneal cavitiesof nude mice to produce ascites containing high concentrations (>1mg/ml) of anti-IL-4R monoclonal antibody. The resulting monoclonalantibodies may be purified by ammonium sulfate precipitation followed bygel exclusion chromatography, and/or affinity chromatography based onbinding of antibody to Protein G.

Example 2: Generation of Cmu Targeted Mice

This example describes procedures for generating transgenic mice.Additional procedures for generating transgenic mice, and the use ofsuch mice for preparing human antibodies, are described in Examples 3and 4.

Construction of a CMD Targeting Vector.

The plasmid pICEmu contains an EcoRI/XhoI fragment of the murine Igheavy chain locus, spanning the mu gene, that was obtained from a Balb/Cgenomic lambda phage library (Marcu et al. Cell 22: 187, 1980). Thisgenomic fragment was subcloned into the XhoI/EcoRI sites of the plasmidpICEMI9H (Marsh et al; Gene 32, 481-485, 1984). The heavy chainsequences included in pICEmu extend downstream of the EcoRI site locatedjust 3′ of the mu intronic enhancer, to the XhoI site locatedapproximately 1 kb downstream of the last transmembrane exon of the mugene; however, much of the mu switch repeat region has been deleted bypassage in E. coli.

The targeting vector was constructed as follows. (See FIGS. 2A-2C, whichdepict further details.) A 1.3 kb HindIII/SmaI fragment was excised frompICEmu and subcloned into HindIII/SmaI digested pBluescript (Stratagene,La Jolla, Calif.). This pICEmu fragment extends from the HindIII sitelocated approximately 1 kb 5′ of Cmu1 to the SmaI site located withinCmu1. The resulting plasmid was digested with SmaI/SpeI and theapproximately 4 kb SmaI/XbaI fragment from pICEmu, extending from theSma I site in Cmu1 3′ to the XbaI site located just downstream of thelast Cmu exon, was inserted. The resulting plasmid, pTAR1, waslinearized at the SmaI site, and a neo expression cassette inserted.This cassette consists of the neo gene under the transcriptional controlof the mouse phosphoglycerate kinase (pgk) promoter (XbaI/TaqI fragment;Adra et al. (1987) Gene 60: 65-74) and containing the pgkpolyadenylation site (PvuII/HindIII fragment; Boer et al. (1990)Biochemical Genetics 28: 299-308). This cassette was obtained from theplasmid pKJ1 (described by Tybulewicz et al. (1991) Cell 65: 1153-1163)from which the neo cassette was excised as an EcoRI/HindIII fragment andsubcloned into EcoRI/HindIII digested pGEM-7Zf (+) to generate pGEM-7(KJ1). The neo cassette was excised from pGEM-7 (KJ1) by EcoRI/SalIdigestion, blunt ended and subcloned into the SmaI site of the plasmidpTAR1, in the opposite orientation of the genomic Cmu sequences.

The resulting plasmid was linearized with Not I, and a herpes simplexvirus thymidine kinase (tk) cassette was inserted to allow forenrichment of ES clones bearing homologous recombinants, as described byMansour et al. (1988) Nature 336: 348-352. This cassette consists of thecoding sequences of the tk gene bracketed by the mouse pgk promoter andpolyadenylation site, as described by Tybulewicz et al. (1991) Cell65:1153-1163.

The resulting CMD targeting vector contains a total of approximately 5.3kb of homology to the heavy chain locus and is designed to generate amutant mu gene into which has been inserted a neo expression cassette inthe unique SmaI site of the first Cmu exon. The targeting vector waslinearized with PvuI, which cuts within plasmid sequences, prior toelectroporation into ES cells.

Generation and Analysis of Targeted ES Cells.

AB-1 ES cells (McMahon, A. P. and Bradley, A., (1990) Cell 62:1073-1085) were grown on mitotically inactive SNL76/7 cell feeder layers(ibid.), essentially as described in Teratocarcinomas and Embryonic StemCells: a Practical Approach, E. J. Robertson, Ed., Oxford: IRL Press,1987, pp. 71-112. The linearized CMD targeting vector was electroporatedinto AB-1 cells by the methods described in Hasty et al. (1991) Nature350: 243-246. Electroporated cells were plated into 100 mm dishes at adensity of 1-2×10⁶ cells/dish. After 24 hours, G418 (200 micrograms/mlof active component) and FIAU (5×10⁻⁷ M) were added to the medium, anddrug-resistant clones were allowed to develop over 8-9 days. Clones werepicked, trypsinized, divided into two portions, and further expanded.Half of the cells derived from each clone were then frozen and the otherhalf analyzed for homologous recombination between vector and targetsequences.

DNA analysis was carried out by Southern blot hybridization. DNA wasisolated from the clones as described by Laird et al., (1991) NucleicAcids Res. 19:4293). Isolated genomic DNA was digested with SpeI andprobed with a 915 bp SacI fragment, probe A (FIG. 2C), which hybridizesto a sequence between the mu intronic enhancer and the mu switch region.Probe A detects a 9.9 kb SpeI fragment from the wild type locus, and adiagnostic 7.6 kb band from a mu locus which has homologously recombinedwith the CMD targeting vector (the neo expression cassette contains aSpeI site).

Of 1132 G418 and FIAU resistant clones screened by Southern blotanalysis, 3 displayed the 7.6 kb Spe I band indicative of homologousrecombination at the mu locus. These 3 clones were further digested withthe enzymes BglI, BstXI, and EcoRI to verify that the vector integratedhomologously into the mu gene. When hybridized with probe A, Southernblots of wild type DNA digested with BglI, BstXI, or EcoRI producefragments of 15.7, 7.3, and 12.5 kb, respectively, whereas the presenceof a targeted mu allele is indicated by fragments of 7.7, 6.6, and 14.3kb, respectively. All 3 positive clones detected by the SpeI digestshowed the expected BglI, BstXI, and EcoRI restriction fragmentsdiagnostic of insertion of the neo cassette into the Cmu1 exon.

Generation of Mice Bearing the Mutated Mu Gene.

The three targeted ES clones, designated number 264, 272, and 408, werethawed and injected into C57BL/6J blastocysts as described by A. Bradleyin Teratocarcinomas and Embryonic Stem Cells: a Practical Approach, E.J. Robertson, Ed., Oxford: IRL Press, 1987, pp. 113-151. Injectedblastocysts were transferred into the uteri of pseudopregnant females togenerate chimeric mice representing a mixture of cells derived from theinput ES cells and the host blastocyst. The extent of ES cellcontribution to the chimera can be visually estimated by the amount ofagouti coat coloration, derived from the ES cell line, on the blackC57BL/6J background. Clones 272 and 408 produced only low percentagechimeras (i.e. low percentage of agouti pigmentation) but clone 264produced high percentage male chimeras. These chimeras were bred withC57BL/6J females and agouti offspring were generated, indicative ofgermline transmission of the ES cell genome. Screening for the targetedmu gene was carried out by Southern blot analysis of BglI digested DNAfrom tail biopsies (as described above for analysis of ES cell DNA).Approximately 50% of the agouti offspring showed a hybridizing BglI bandof 7.7 kb in addition to the wild type band of 15.7 kb, demonstrating agermline transmission of the targeted mu gene.

Analysis of Transgenic Mice for Functional Inactivation of Mu Gene.

To determine whether the insertion of the neo cassette into Cmu1 hasinactivated the Ig heavy chain gene, a clone 264 chimera was bred with amouse homozygous for the JHD mutation, which inactivates heavy chainexpression as a result of deletion of the JH gene segments (Chen et al,(1993) Immunol. 5: 647-656). Four agouti offspring were generated. Serumwas obtained from these animals at the age of 1 month and assayed byELISA for the presence of murine IgM. Two of the four offspring werecompletely lacking IgM (Table 1). Genotyping of the four animals bySouthern blot analysis of DNA from tail biopsies by BglI digestion andhybridization with probe A (FIG. 2C), and by StuI digestion andhybridization with a 475 bp EcoRI/StuI fragment (ibid.) demonstratedthat the animals which fail to express serum IgM are those in which oneallele of the heavy chain locus carries the JHD mutation, the otherallele the Cmu1 mutation. Mice heterozygous for the JHD mutation displaywild type levels of serum Ig. These data demonstrate that the Cmu1mutation inactivates expression of the mu gene.

Table 1 presents the level of serum IgM, detected by ELISA, for micecarrying both the CMD and JHD mutations (CMD/JHD), for mice heterozygousfor the JHD mutation (+/JHD), for wild type (129Sv×C57BL/6J)F1 mice(+/+), and for B cell deficient mice homozygous for the JHD mutation(JHD/JHD).

TABLE 1 Mouse Serum IgM (micrograms/ml) Ig H chain genotype 42 <0.002CMD/JHD 43 196 +/JHD 44 <0.002 CMD/JHD 45 174 +/JHD 129 × BL6 F1 153 +/+JHD <0.002 JHD/JHD

Example 3: Generation of Transgenic Mice

The HCo12 Human Heavy Chain Transgene.

The HCo12 transgene was generated by coinjection of the 80 kb insert ofpHC2 (Taylor et al., 1994, Int. Immunol., 6: 579-591) and the 25 kbinsert of pVx6. The plasmid pVx6 was constructed as described below.

An 8.5 kb HindIII/SalI DNA fragment, comprising the germline humanVH1-18 (DP-14) gene together with approximately 2.5 kb of 5′ flanking,and 5 kb of 3′ flanking genomic sequence was subcloned into the plasmidvector pSP72 (Promega, Madison, Wis.) to generate the plasmid p343.7.16.A 7 kb BamHI/HindIII DNA fragment, comprising the germline human VH5-51(DP-73) gene together with approximately 5 kb of 5′ flanking and 1 kb of3′ flanking genomic sequence, was cloned into the pBR322 based plasmidcloning vector pGP1f (Taylor et al. 1992, Nucleic Acids Res. 20:6287-6295), to generate the plasmid p251f.

A new cloning vector derived from pGP1f, pGP1k (the sequence of which ispresented in FIGS. 3A and 3B and SEQ ID NO:4), was digested withEcoRV/BamHI, and ligated to a 10 kb EcoRV/BamHI DNA fragment, comprisingthe germline human VH3-23 (DP47) gene together with approximately 4 kbof 5′ flanking and 5 kb of 3′ flanking genomic sequence. The resultingplasmid, p112.2RR.7, was digested with BamHI/SalI and ligated with the 7kb purified BamHI/SalI insert of p251f. The resulting plasmid, pVx4, wasdigested with XhoI and ligated with the 8.5 kb XhoI/SalI insert ofp343.7.16. A clone was obtained with the VH1-18 gene in the sameorientation as the other two V genes. This clone, designated pVx6, wasthen digested with NotI and the purified 26 kb insert coinjected,together with the purified 80 kb NotI insert of pHC2 at a 1:1 molarratio, into the pronuclei of one-half day (C57BL/6J×DBA/2J)F2 embryos asdescribed by Hogan et al. (B. Hogan et al., Manipulating the MouseEmbryo, A Laboratory Manual, 2^(nd) edition, 1994, Cold Spring HarborLaboratory Press, Plainview N.Y.).

Three independent lines of transgenic mice comprising sequences fromboth Vx6 and HC2 were established from mice that developed from theinjected embryos. These lines are designated (HCo12)14881, (HCo12)15083,and (HCo12)15087. Each of the three lines were then bred with micecomprising the CMD mutation described in Example 2, the JKD mutation(Chen et al. 1993, EMBO J. 12: 811-820), and the (KCo5)9272 transgene(Fishwild et al. 1996, Nature Biotechnology 14: 845-851). The resultingmice express human heavy and kappa light chain transgenes in abackground homozygous for disruption of the endogenous mouse heavy andkappa light chain loci.

Additional Transgenic Mouse Strains

Particular strains of mice that may be used to generate IL-4R-reactivemonoclonal antibodies are strain ((CMD)++; (JKD)++; (HCo7) 11952+/++;(KCo5)9272+/++), and strain ((CMD)++; (JKD)++; (HCo12)15087+/++;(KCo5)9272+/++). Each of these transgenic strains is homozygous fordisruptions of the endogenous heavy chain (CMD) and kappa light chain(JKD) loci. Both strains also comprise a human kappa light chaintransgene (HCo7), with individual animals either hemizygous orhomozygous for insertion #11952. The two strains differ in the humanheavy chain transgene used. Mice were hemizygous or homozygous foreither the HCo7 or the HCo12 transgene. The CMD mutation is describedabove in Example 2. The generation of (HCo12)15087 mice is describedabove (in this example). The JKD mutation (Chen et al. 1993, EMBO J. 12:811-820) and the (KCo5)9272 (Fishwild et al. 1996, Nature Biotechnology14: 845-851) and (HCo7)11952 mice, are described in U.S. Pat. No.5,770,429, which is hereby incorporated by reference.

Example 4: Generation of Human Anti-IL-4R Monoclonal Antibodies

Transgenic Mice

Strain ((CMD)++; (JKD)++; (HCo7)11952+/++; (KCo5)9272+/++ which ishomozygous for disruptions of the endogenous heavy chain (CMD) and kappalight chain (JKD) loci (see example 3), was used to generateIL-4R-reactive monoclonal antibodies. This strain also comprises a humankappa light chain transgene (HCo7) with individual animals eitherhemizygous or homozygous for insertion #11952. Mice were hemizygous orhomozygous for the HCo7 transgene. The CMD mutation is described abovein Example 2. The JKD mutation (Chen et al. 1993, EMBO J. 12: 811-820)and the (KCo5)9272 (Fishwild et al. 1996, Nature Biotechnology 14:845-851) and (HCo7)11952 mice, are described in U.S. Pat. No. 5,770,429,which is hereby incorporated by reference.

Immunization.

Transgenic mice were initially immunized i.p. with 25 ug IL-4R proteinin adjuvant (Titermax, available from Cytrx Corporation, Norcross, Ga.).The immunogen was a human IL-4R polypeptide comprising the extracellulardomain of the protein of SEQ ID NO:2. Immunized mice were subsequentlyboosted every 4 weeks i.p. with the IL-4R immunogen in incompletefreunds adjuvant. Animals were kept on protocol for 2 to 5 months. Priorto fusion, animals were boosted i.v. on days −4 and −3 with 5 to 8 ugimmunogen.

Fusions.

Spleen cells harvested from the immunized mice were fused to mousemyeloma cells NS-1 by standard procedures (Harlow and Lane, 1988,Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor N.Y.; Kennett et al. 1980, Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analysis. Plenum, N.Y.; Oi andHertzenberg, 1980, Immunoglobulin Producing Hybrid Cell Lines, inSelected Methods In Cellular Immunology, ed. Mishell and Shiigi, pp.357-372. Freeman, San Francisco). Cells were cultured in DMEM, 10% FBS,OPI (Sigma 0-5003), BME (Gibco 21985-023), 3% Origen Hybridoma CloningFactor (Igen IG50-0615), and 5% P388d1 (ATCC TIB 63) conditioned media.HAT or HT supplement was added to the medium during initial growth andselection.

Hybridoma Screening.

To identify hybridomas secreting human antibodies against the IL-4R,ELISA plates (Nunc MaxiSorp) were coated overnight at 4° C. with 100ul/well human IL-4R at 2.0 ug/ml in PBS. Plates were washed with 100ul/well PBS-Tween (PBST) containing 1% BSA. Fifty ul cell culturesupernatent was added followed by a 1.0 hour incubation. Plates werewashed and then incubated for one hour with 100 ul/well goat anti-humanIgG conjugated to horseradish peroxidase (Sigma #A-3813, or #A-7164).Plates were washed three times in PBS-Tween between each step.

Wells that read positive by ELISA were screened for their ability toblock the binding of IL-4 to IL-4R. ELISA plates were coated overnightwith a non-neutralizing mouse anti-human IL-4R antibody M10 at 2 ug/ml.Plates were washed 3× with PBST. 100 ul human IL-4R was added at 10ng/ml in PBST and incubated for 1.0 hour. Plates were washed 4× withPBST and 100 ul supernatant samples were added and incubated for 1.0hour. Wells were washed 4× with PBST. 5.0 ng/ml biotinylated IL-4 wasadded in PBST and incubated for 1.0 hour. 100 ul/well poly80 horseradishperoxidase (RDI) was added at 1:5000 in PBST and incubated for 45minutes. Plates were washed 5× with PBST, and a colorimetric reagent(3,3′,5,5′ tetramethylbenzidine, available from Kirkegaard and Perry)was added at 100 ul/well until color developed. Reaction was stoppedwith 100 ul phosphoric acid and plates were read at 450 nm. Absent orreduced signal was interpreted as the antibody binding to receptor in amanner that blocked IL-4 from binding to receptor. Wells that appearedto block binding were expanded and tested for IL-4 and IL-13 blocking ina CD23 expression assay (see example 5).

Example 5: Assay for Assessing Blocking Activity

This assay is based on ability of both IL-4 and IL-13 to enhance theexpression of the activation-associated surface antigen CD23 on human Bcells. Antibodies are tested for the ability to inhibit CD23 expressioninduced by IL-4 and by IL-13.

Antibodies raised against human IL-4R (huIL-4R) were tested either inthe form of hybridoma supernatants or purified protein. Prior toaddition to cultures, the antibodies were buffer exchanged againstculture medium (RPMI 1640 plus 10% heat-inactivated fetal bovine serum)by centrifugation, using Centricon filter devices (Amicon) with a 10 kDacutoff.

Human peripheral blood B cells were purified as described previously(Morris et al., J. Biol. Chem. 274:418-423, 1999). The B cells(3×10⁵/well) in culture medium were placed in 96-well round-bottomedmicrotiter plates and preincubated at room temperature for 30 min withtest antibodies at the final concentrations indicated. Recombinant humanIL-4 or IL-13 was then added to the cultures at the concentrationsindicated, and cells were cultured for 20-24 hours at 37° C. in ahumidified atmosphere of 5% CO₂. At the end of the culture period, cellswere washed once in PBS+0.02% NaN₃ in the 96-well culture plate and wereresuspended in blocking buffer (2% normal rabbit serum+1% normal goatserum in PBS+NaN₃). Phycoerythrin (PE)-conjugated CD23 monoclonalantibody (mAb) or PE-conjugated isotype control mAb (both fromPharmingen) was then added to cells at a final dilution of 1:10. Cellswere incubated for 30 minutes at 4° C., washed ×3 in PBS+NaN₃ andanalyzed on a FacScan (Becton Dickinson) for CD23 expression.

In all experiments, negative controls were included which consisted ofcells cultured with hybridoma growth medium or isotype-matchednon-blocking human anti-hIL-4R antibody. An anti-huIL-4R murine mAb (R&DSystems), previously shown to block the binding and function of bothhIL-4 and hIL-13, was used as a positive control for neutralization ofCD23 induction by IL-4 and IL-13.

Example 6: Hybridoma Cell Line

One hybridoma cell line generated by procedures described above (seeexample 4) is designated 6-2. The anti-IL-4R monoclonal antibodysecreted by this hybridoma is a blocking antibody, as determined in aconventional plate binding assay, and thus functions as an IL-4antagonist. The monoclonal antibody produced by 6-2 also exhibits theability to reduce an IL-13-induced biological activity.

One embodiment of the invention is directed to a hybridoma cell lineproduced as described above, wherein the hybridoma secretes an isotypeIgM MAb directed against human IL-4R. Also provided herein are IgG1monoclonal antibodies derived from IgM monoclonal antibodies.

The DNA sequence of the variable region of the light chain of MAb 6-2has been determined, and is presented in SEQ ID NO:5; the amino acidsequence encoded thereby is presented in SEQ ID NO:6. Complementaritydetermining regions 1 to 3 (CDR 1-3) are believed to correspond to aminoacids 24-35, 51-57, and 90-97, of SEQ ID NO:6, respectively.

The DNA sequence of the variable region of the heavy chain of MAb 6-2has been determined, and is presented in SEQ ID NO:7; the amino acidsequence encoded thereby is presented in SEQ ID NO:8. Complementaritydetermining regions 1 to 3 (CDR 1-3) are believed to correspond to aminoacids 31-35, 50-66, and 99-107 of SEQ ID NO:8, respectively.

Example 7: Assays for Measuring Loss of Barrier Function

A method provided herein involves use of IL-4 antagonists to inhibitIL-4-induced damage to epithelium, including but not limited to lungepithelium or intestinal epithelium. Damage to epithelium can result inloss of barrier function. A number of techniques are known fordetermining whether an epithelial layer is intact. The following areexamples of techniques that may be employed in assessing the ability ofan IL-4 antagonist to inhibit IL-4-induced damage to epithelium and lossof epithelial barrier function.

Cells that may be employed in preparing in vitro models of epithelium(epithelial barriers) are known. For example, Calu-3 human lungepithelial cells are suitable for use in barrier function studies.Another suitable cell line is the human intestinal epithelial cell linedesignated T84. T84 cells are cultured under conditions that result information of a monolayer of epithelial cells on a permeable support, asdescribed in Madara, J. and K. Dharmsathaphorn (J. Cell Biol.,101:2124-2133, 1985), Madara, J. and J. Stafford (J. Clin. Invest.83:724-727, 1989), and Youakim, A. and M. Ahdieh (Am. J. Physiol. 276(Gastrointest. Liver Physiol. 39):G1279-G1288, 1999). The culturedmonolayers are tested for properties such as resistance to passivetransepithelial ion flow (such resistance indicating an intact monolayerperforming a barrier function). The thus-generated epithelial monolayersimulates the intestinal epithelial barrier.

One type of assay determines whether a particular radiolabeled compoundis able to cross an epithelial monolayer (e.g., a monolayer generated asdescribed above). Transport of the radiolabeled compound across themonolayer indicates that the barrier is permeable rather than intact.One such procedure is mannitol flux analysis, which assesses movement ofradiolabeled mannitol (e.g., ³H mannitol) across a monolayer (see Madaraand Stafford, supra).

Methods for imaging a monolayer are identified in Madara and Stafford,supra. Such imaging methods are an alternative for assessing thecondition of an epithelial layer, after exposure to IL-4 with or withoutan antagonist.

Youakim and Ahdieh, supra, discuss proteins that are part of “tightjunction” complexes in intact intestinal epithelial barriers, and reportstudies of the effect of IFN-γ on proteins associated with tightjunctions. Other techniques for studying the effect of a cytokine onbarrier function are described. For example, the effect of a cytokine onmonolayer permeability may be assessed by transepithelial electricalresistance measurements, using techniques described in the reference.

U.S. Pat. No. 6,033,688 also describes procedures that may be employedin studies of barrier permeability; see especially examples 1 and 4 ofthe patent. Human tracheal epithelial cells were cultured underconditions that yielded a monolayer exhibiting transepithelialelectrical resistance. Transepithelial resistance (indicating an intactbarrier) was determined using a voltmeter. The effect of a particularreagent (HGH) on the epithelial monolayer was assessed by exposing themonolayer to HGH, and then measuring ion transport activities in Ussingchambers, by standard methods (column 8, lines 40-56). Similar studieswere conducted on monolayers that were generated from bronchialepithelial cells from a human cystic fibrosis patient (example 4, column11).

Using any of the above-described barrier function assay procedures, anepithelial monolayer is exposed to IL-4 alone, or exposed to IL-4 in thepresence of an IL-4 antagonist. The antagonist's ability to inhibit theIL-4-induced reduction in barrier function thus is assessed.

In one such assay, a monolayer of T84 cells served as an in vitro modelof an intestinal epithelial barrier, as discussed above. IL-4 added tothe basolateral side of polarized epithelial cells was found to reducebarrier function by 70% within 48-72 hours of treatment. When an IL-4receptor polypeptide was added at the same time as IL-4, the reductionin barrier function was prevented, and the barrier was maintained at thesame level as untreated (control) cells. A soluble human IL-4 receptorpolypeptide, consisting of the extracellular domain, was employed in theassay.

The assay procedure also was conducted on a monolayer derived from lungepithelial cells, which served as an in vitro model of a lung epithelialbarrier. IL-4 added to the basolateral side of polarized lung epithelialcells was found to reduce barrier function by 50% within 48-72 hours oftreatment. When the IL-4 receptor polypeptide was added at the same timeas IL-4, the reduction in barrier function was prevented, and thebarrier was maintained at the same level as untreated (control) cells.

Example 8: Monoclonal Antibody Designated 12B5

A human monoclonal antibody directed against human IL-4 receptor wasprepared by the following procedure. The monoclonal antibody, which isdesignated 12B5, is a blocking antibody that functions as an IL-4antagonist and as an IL-13 antagonist.

The procedure began with immunization of a transgenic mouse with asoluble human IL-4 receptor polypeptide. Mouse strain ((CMD)++; (JKD)++;(HCo7)11952+/++; (KCo5)9272+/++), described in example 3 above, wasemployed. The antigen for immunization was purified soluble IL-4R,comprising the extracellular domain of human IL-4 receptor (500 ug/ml).The mouse was initially immunized with 50 ug antigen emulsified inComplete Freund's Adjuvant, followed by two more immunizations withIncomplete Freund's Adjuvant at 50 ug and then 25 ug. Immunization wasevery two weeks by intraperitoneal injection. A specific human IgGanti-IL-4R titer from the serum was performed by ELISA, eight days afterthe last injection. A good titer against the target was detected, andthe mouse then was IV/IP boosted 25 ug each on day ⁻3 (i.e., 3 daysbefore the mouse was sacrificed) and 15 ug IV on day ⁻2.

The mouse was sacrificed, and spleen cells were extracted and fused withthe murine myeloma cell line P3x63Ag8.653 (ATCC CRL 1580). Aconventional PEG fusion protocol was followed. The fusion was screenedfor HuIgG and HuKappa, followed by a rescreen for human gamma and kappaspecific to IL-4R. The positive clones were evaluated, and clone 12B5was identified as a blocking antibody. The clone was subcloned; ahybridoma cell line that produces MAb 12B5 was isolated; and MAb 12B5was purified from the supernatant.

12B5 was determined to be an IgG1 antibody, and to be fully human.Antibodies of other subclasses, such as IgG4 or IgM monoclonalantibodies, may be derived from 12B5. Techniques for altering(switching) the subclass/isotype of an antibody are known. The constantregion of 12B5 may be replaced, for example, with a constant regionderived from a human IgG4 antibody. Sequence information for a humanIgG4 heavy chain is presented, for example, in Ellison et al. (DNA Vol.1, no. 1, pp 11-18, 1981), which is hereby incorporated by referenceherein.

DNA encoding the variable region of the light chain of MAb 12B5 wasisolated, and the nucleotide sequence thereof was determined. The DNAsequence for the light chain variable region is presented as SEQ IDNO:9; the amino acid sequence encoded thereby is presented in SEQ IDNO:10. Complementarity determining regions 1 to 3 (CDR 1-3) are believedto correspond to amino acids 24-35, 51-57, and 90-99, of SEQ ID NO:10,respectively.

DNA encoding the variable region of the heavy chain of MAb 12B5 wasisolated, and the nucleotide sequence thereof was determined. The DNAsequence for the heavy chain variable region is presented as SEQ IDNO:11; the amino acid sequence encoded thereby is presented in SEQ IDNO:12. CDR-1 of the heavy chain is believed to correspond to amino acids31-35; CDR-2 to amino acids 50-65; and CDR-3 to amino acids 98-104 ofSEQ ID NO:12.

Example 9: Additional Monoclonal Antibodies that Inhibit Both IL-4 andIL-13

Additional human monoclonal antibodies were raised against human IL-4receptor, by immunizing transgenic mice with a soluble human IL-4Rpolypeptide. The transgenic mice employed were selected from thetransgenic mouse strains described in example 3.

Hybridoma cell lines secreting human monoclonal antibodies thatspecifically bind human IL-4R, and which are capable of functioning asIL-4 antagonists and IL-13 antagonists, were identified and isolated.The MAbs are designated 27A1, 5A1, and 63. Another MAb, designated 1B7,was derived from MAb 63, and differs from the parent antibody only inthe light chain. 1B7 retains the ability to bind IL-4R and to functionas an IL-4 antagonist and an IL-13 antagonist.

The DNA sequence of the variable region of the light chain of MAb 27A1is presented in SEQ ID NO:13, and the encoded amino acid sequence ispresented in SEQ ID NO:14. Complementarity determining regions 1 to 3(CDR 1-3) are believed to correspond to amino acids 24-35, 51-57, and90-99, of SEQ ID NO:14, respectively.

The DNA sequence for the variable region of the heavy chain of MAb 27A1is presented as SEQ ID NO:15, and the encoded amino acid sequence ispresented in SEQ ID NO:16. Complementarity determining regions 1 to 3(CDR 1-3) are believed to correspond to amino acids 31-35, 50-66, and99-105, of SEQ ID NO:16, respectively.

The DNA sequence of the variable region of the light chain of MAb 5A1 ispresented in SEQ ID NO:17, and the encoded amino acid sequence ispresented in SEQ ID NO:18. Complementarity determining regions 1 to 3(CDR 1-3) are believed to correspond to amino acids 24-34, 50-56, and89-97 of SEQ ID NO:18, respectively.

The DNA sequence for the variable region of the heavy chain of MAb 5A1is presented as SEQ ID NO:19, and the encoded amino acid sequence ispresented in SEQ ID NO:20. Complementarity determining regions 1 to 3(CDR 1-3) are believed to correspond to amino acids 31-35, 50-65, and98-112 of SEQ ID NO:20, respectively.

The DNA sequence of the variable region of the light chain of MAb 63 ispresented in SEQ ID NO:21, and the encoded amino acid sequence ispresented in SEQ ID NO:22. Complementarity determining regions 1 to 3(CDR 1-3) are believed to correspond to amino acids 24-34, 50-56, and89-97 of SEQ ID NO:22, respectively.

The DNA sequence for the variable region of the heavy chain of MAb 63 ispresented as SEQ ID NO:23, and the encoded amino acid sequence ispresented in SEQ ID NO:24. Complementarity determining regions 1 to 3(CDR 1-3) are believed to correspond to amino acids 31-35, 50-66, and99-106 of SEQ ID NO:24, respectively.

The DNA sequence of the variable region of the light chain of MAb 1B7 ispresented in SEQ ID NO:25, and the encoded amino acid sequence ispresented in SEQ ID NO:26. Complementarity determining regions 1 to 3(CDR 1-3) are believed to correspond to amino acids 24-34, 50-56, and89-97 of SEQ ID NO:26, respectively.

MAb 1B7 was derived from MAb 63, and the heavy chains of the two MAbsare identical. Thus, the DNA sequence for the variable region of theheavy chain of MAb 1B7 is presented as SEQ ID NO:23, and the encodedamino acid sequence is presented in SEQ ID NO:24. Complementaritydetermining regions 1 to 3 (CDRs 1-3) are believed to correspond toamino acids 31-35, 50-66, and 99-106 of SEQ ID NO:24, respectively.

What is claimed is:
 1. An isolated anti-IL4 receptor antibody comprisinga light-chain variable region comprising the amino acid sequence of SEQID NO:6 and a heavy-chain variable region comprising the amino acidsequence of SEQ ID NO:8.
 2. The isolated antibody of claim 1, whereinsaid antibody: a) antagonizes IL-4; or b) antagonizes IL-13; or c)antagonizes IL-4 and IL-13.
 3. The isolated antibody of claim 1, whereinsaid antibody is a monoclonal antibody.
 4. The isolated antibody ofclaim 1, wherein said antibody is an IgG antibody.
 5. A nucleic acidthat encodes the light-chain, the heavy-chain, or the light-chain andthe heavy-chain of the antibody of claim
 1. 6. An isolated cellcomprising the nucleic acid of claim
 5. 7. The isolated cell of claim 6,wherein said cell is a hybridoma.
 8. The isolated cell of claim 6,wherein said cell is a recombinant cell.
 9. A method of manufacturing anantibody, comprising incubating the isolated cell of claim 6 underconditions that allow it to express an anti-IL4 receptor antibodycomprising a light-chain variable region comprising the amino acidsequence of SEQ ID NO:6 and a heavy-chain variable region comprising theamino acid sequence of SEQ ID NO:8 and isolating said antibody.
 10. Amethod of treating an allergic disease, comprising administering saidantibody of claim 1 to a patient with said allergic disease.
 11. Themethod of claim 10, wherein said allergic disease is allergic rhinitis,asthma, atopic dermatitis, or contact dermatitis.